U.S. patent application number 13/257418 was filed with the patent office on 2012-04-26 for compounds and methods for treating mammalian gastrointestinal microbial infections.
This patent application is currently assigned to University of Georgia Foundation, Inc.. Invention is credited to Gregory D. Cuny, Deviprasad R. Gollapalli, Suresh Kumar Gorla, Lizbeth K. Hedstrom, Corey Robert Johnson, Mandapati Kavitha, Jihan Khan, Sivapriya Kirubakaran, Sushil Kumar Maurya, Boris Striepen.
Application Number | 20120101096 13/257418 |
Document ID | / |
Family ID | 42740276 |
Filed Date | 2012-04-26 |
United States Patent
Application |
20120101096 |
Kind Code |
A1 |
Hedstrom; Lizbeth K. ; et
al. |
April 26, 2012 |
Compounds and Methods for Treating Mammalian Gastrointestinal
Microbial Infections
Abstract
Described herein are compounds, and pharmaceutically acceptable
salts and prodrugs thereof, which are useful as inhibitors of
IMPDH. In certain embodiments, a compound of the invention
selectively inhibits a parasitic IMPDH versus a host IMPDH.
Further, the invention provides pharmaceutical compositions
comprising one or more compounds of the invention. The invention
also relates to methods of treating various parasitic and bacterial
infections in mammals. Moreover, the compounds may be used alone or
in combination with other therapeutic or prophylactic agents, such
as anti-virals, anti-inflammatory agents, antimicrobials and
immunosuppressants.
Inventors: |
Hedstrom; Lizbeth K.;
(Newton, MA) ; Cuny; Gregory D.; (Somerville,
MA) ; Gollapalli; Deviprasad R.; (Waltham, MA)
; Kirubakaran; Sivapriya; (Chennai, IN) ; Maurya;
Sushil Kumar; (Leeds, GB) ; Striepen; Boris;
(Athens, GA) ; Gorla; Suresh Kumar; (Waltham,
MA) ; Johnson; Corey Robert; (Waltham, MA) ;
Kavitha; Mandapati; (Waltham, MA) ; Khan; Jihan;
(Somerville, MA) |
Assignee: |
University of Georgia Foundation,
Inc.
Athens
GA
Brandeis University
Waltham
MA
|
Family ID: |
42740276 |
Appl. No.: |
13/257418 |
Filed: |
March 22, 2010 |
PCT Filed: |
March 22, 2010 |
PCT NO: |
PCT/US10/28178 |
371 Date: |
December 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61162013 |
Mar 20, 2009 |
|
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Current U.S.
Class: |
514/234.5 |
Current CPC
Class: |
A61P 35/00 20180101;
C07C 275/28 20130101; C07D 401/12 20130101; C07D 491/052 20130101;
C07C 235/24 20130101; C07D 209/08 20130101; C07D 471/08 20130101;
C07D 235/26 20130101; C07C 275/42 20130101; C07D 417/04 20130101;
C07D 401/04 20130101; C07C 237/20 20130101; C07D 209/30 20130101;
C07D 417/14 20130101; C07D 409/04 20130101; C07D 413/04 20130101;
C07D 263/56 20130101; C07C 309/15 20130101; C07D 295/192 20130101;
C07C 235/38 20130101; C07C 275/30 20130101; C07D 237/32 20130101;
A61P 19/02 20180101; A61P 33/00 20180101; C07D 263/57 20130101;
C07D 401/14 20130101; C07D 215/38 20130101; C07C 233/15 20130101;
C07C 255/60 20130101; C07D 209/12 20130101; A61P 37/00 20180101;
C07D 249/06 20130101 |
Class at
Publication: |
514/234.5 |
International
Class: |
A61K 31/535 20060101
A61K031/535 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was made with support provided by the National
Institutes of Health (Grant Nos. U01 AI-75466); therefore, the
government has certain rights in the invention.
Claims
1. A compound, or a pharmaceutically acceptable salt thereof,
represented by Formula I: ##STR00256## wherein, independently for
each occurrence, R.sup.1 is hydrogen, alkyl, aryl, heteroaryl,
aralkyl, or heteroaralkyl; R.sup.2 is hydrogen or alkyl; or R.sup.1
and an instance of R.sup.2 taken together with the carbon atoms to
which they are attached form a 5-, 6-, or 7-membered aryl or
heteroaryl ring; R.sup.3 is hydrogen or alkyl; Y.sup.1 is absent,
O, or NR.sup.4; Y.sup.2 is absent, O, NR.sup.4, alkylene,
--(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--,
--(CH.sub.2).sub.m--NR.sup.4--(CH.sub.2).sub.p--,
--(CH.sub.2).sub.m--C(.dbd.O)--(CH.sub.2).sub.p--,
--(CH.sub.2).sub.m--C(.dbd.O)NR.sup.4--(CH.sub.2).sub.p--, or
--(CH.sub.2).sub.mC(.dbd.O)O--(CH.sub.2).sub.p--; n is 0, 1, 2, 3,
or 4; ##STR00257## is aryl or heteroaryl; ##STR00258## is hydrogen,
aryl, or heteroaryl; R.sup.4 is hydrogen or alkyl; m is 0, 1, 2, 3,
or 4; and p is 0, 1, or 2; wherein, any of the aforementioned
alkyl, aryl, heteroaryl, or aralkyl may be substituted with one or
more groups independently selected from the group consisting of
halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy,
alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,
acyloxy, silyl, alkylthio, sulfonate, sulfonyl, sulfonamido,
formyl, cyano, and isocyano.
2. A compound, or a pharmaceutically acceptable salt thereof,
represented by Formula VI: ##STR00259## wherein, independently for
each occurrence, R.sup.1 is hydrogen, alkyl, cycloalkyl, aryl,
heteroaryl, aralkyl, or heteroaralkyl; R.sup.2 is hydrogen or
alkyl; R.sup.3 is hydrogen or alkyl; n is 0, 1, 2, 3, or 4; X is
absent, alkylene, --NR.sup.3--, --SO.sub.2--, or
--CR.sup.3.dbd.N--; Z is --N.dbd. or --CR.sup.5.dbd.; ##STR00260##
is aryl or heteroaryl; ##STR00261## is selected from the group
consisting of hydrogen, alkyl, aryl, and heteroaryl; q is 0, 1, 2,
3, or 4; and R.sup.5 is halo, azido, alkyl, haloalkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
heteroaralkyl, hydroxy, alkoxy, haloalkyloxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, alkylthio,
sulfonate, sulfonyl, sulfonamido, formyl, cyano, or isocyano;
wherein, any of the aforementioned alkyl, aryl, heteroaryl, or
aralkyl may be substituted with one or more groups independently
selected from the group consisting of halo, azido, alkyl,
haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, silyl,
alkylthio, sulfonate, sulfonyl, sulfonamido, formyl, cyano, and
isocyano.
3. A compound, or a pharmaceutically acceptable salt thereof,
represented by Formula IX: ##STR00262## wherein, independently for
each occurrence, R.sup.2 is hydrogen or alkyl; m is 0, 1, or 2;
##STR00263## is aryl, heteroaryl, amino, alkyl, cycloalkyl,
heterocycloalkyl, or aralkyl; wherein, any of the aforementioned
alkyl, aryl, heteroaryl, or aralkyl may be substituted with one or
more groups independently selected from the group consisting of
halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy,
alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,
acyloxy, silyl, alkylthio, sulfonate, sulfonyl, sulfonamido,
formyl, cyano, and isocyano.
4. A compound, or a pharmaceutically acceptable salt thereof,
represented by Formula X: ##STR00264## wherein, independently for
each occurrence, m is 0, 1, 2, or 3; X is absent, O, S, or NH; and
##STR00265## is aryl or heteroaryl; wherein, any of the
aforementioned aryl or heteroaryl, may be substituted with one or
more groups independently selected from the group consisting of
halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy,
alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,
acyloxy, silyl, alkylthio, sulfonate, sulfonyl, sulfonamido,
formyl, cyano, and isocyano.
5. A compound, or a pharmaceutically acceptable salt thereof,
represented by Formula XI: ##STR00266## wherein, independently for
each occurrence, m is 0, 1, or 2; R.sup.2 is hydrogen or alkyl;
R.sup.3 is hydrogen or alkyl; and ##STR00267## is aryl or
heteroaryl; wherein, any of the aforementioned alkyl, aryl, or
heteroaryl may be substituted with one or more groups independently
selected from the group consisting of halo, azido, alkyl,
haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, silyl,
alkylthio, sulfonate, sulfonyl, sulfonamido, formyl, cyano, and
isocyano.
6. A compound, or a pharmaceutically acceptable salt thereof,
represented by Formula XII: ##STR00268## wherein, independently for
each occurrence, m is 0, 1, or 2; R.sup.2 is hydrogen or alkyl;
##STR00269## is aryl or heteroaryl; and ##STR00270## is aryl or
heteroaryl; wherein, any of the aforementioned alkyl, aryl, or
heteroaryl may be substituted with one or more groups independently
selected from the group consisting of halo, azido, alkyl,
haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, silyl,
alkylthio, sulfonate, sulfonyl, sulfonamido, formyl, cyano, and
isocyano.
7. A compound, or a pharmaceutically acceptable salt thereof,
represented by Formula XIII: ##STR00271## wherein, independently
for each occurrence, X is absent or 0; m is 0, 1, or 2; R.sup.2 is
hydrogen or alkyl; ##STR00272## is hydrogen, aryl, or heteroaryl;
and ##STR00273## is hydrogen, aryl, alkyl, or heteroaryl; wherein,
any of the aforementioned alkyl, aryl, or heteroaryl may be
substituted with one or more groups independently selected from the
group consisting of halo, azido, alkyl, haloalkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
heteroaralkyl, hydroxy, alkoxy, aryloxy, heteroaryloxy, amino,
nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,
carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio, sulfonate,
sulfonyl, sulfonamido, formyl, cyano, and isocyano.
8. A method of killing or inhibiting the growth of a microbe,
comprising the step of contacting said microbe with an effective
amount of a compound represented by any one of Formula I, Formula
VI, Formula IX, Formula X, Formula XI, Formula XII, or Formula
XIII.
9. The method of claim 8, wherein said microbe is a protozoon, a
bacterium, or a fungus.
10. The method of claim 8, wherein said microbe is a protozoon or a
bacterium selected from the group consisting of the genera
Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,
Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,
Tritrichomonas, Leishmania, Trypanosoma, Helicobacter, Borrelia,
Salmonella, Shigella, Yersinia, Streptococcus, Campylobacter,
Arcobacter, Bacteroides, Fusobacterium, Burkholderia, Clostridia,
Neisseria, Mycobacterium, and Acinetobacter.
11. The method of claim 9, wherein said microbe is a protozoon; and
said protozoon is selected from the group consisting of the genera
Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,
Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,
Tritrichomonas, Leishmania and Trypanosoma.
12-13. (canceled)
14. The method of claim 9, wherein said microbe is a bacterium; and
said bacterium is selected from the group consisting of the genera
Helicobacter, Borrelia, Salmonella, Shigella, Yersinia,
Streptococcus, Campylobacter, Arcobacter, Bacteroides,
Fusobacterium, Burkholderia, Clostridia, Neisseria, Mycobacterium,
and Acinetobacter.
15. (canceled)
16. A method of treating or preventing a microbial infection in a
mammal comprising the step of administering to a mammal in need
thereof a therapeutically effective amount of a compound
represented by any one of Formula I, Formula VI, Formula IX,
Formula X, Formula XI, Formula XII, or Formula XIII.
17. The method of claim 16, wherein said microbial infection is
caused by a protozoon, a bacterium, or a fungus.
18. The method of claim 16, wherein said microbial infection is
caused by a protozoon or a bacterium selected from the group
consisting of the genera Toxoplasma, Eimeria, Cryptosporidium,
Plasmodium, Babesia, Theileria, Neospora, Sarcocystis, Giardia,
Entamoeba, Trichomonas, Leishmania, Trypanosoma, Helicobacter,
Borrelia, Salmonella, Shigella, Yersinia, Streptococcus,
Campylobacter, Arcobacter, Bacteroides, Fusobacterium,
Burkholderia, Clostridia, Neisseria, Mycobacterium, and
Acinetobacter.
19. The method of claim 17, wherein said microbial infection is
caused by a protozoon; and said protozoon is selected from the
group consisting of the genera Toxoplasma, Eimeria,
Cryptosporidium, Plasmodium, Babesia, Theileria, Neospora,
Sarcocystis, Giardia, Entamoeba, Trichomonas, Tritrichomonas,
Leishmania and Trypanosoma.
20-21. (canceled)
22. The method of claim 16, wherein said microbial infection is
caused by a bacterium; and said bacterium is selected from the
group consisting of the genera Helicobacter, Borrelia, Salmonella,
Shigella, Yersinia, Streptococcus, Campylobacter, Arcobacter,
Bacteroides, Fusobacterium, Burkholderia, Clostridia, Neisseria,
Mycobacterium, and Acinetobacter.
23-26. (canceled)
27. A method of treating or preventing an IMPDH-mediated disease in
a mammal, comprising the step of administering to a mammal in need
thereof a therapeutically effective amount of a compound
represented by any one of Formula I, Formula VI, Formula IX,
Formula X, Formula XI, Formula XII, or Formula XIII.
28. The method of claim 27, wherein the IMPDH-mediated disease is
transplant rejection, graft versus host disease, rheumatoid
arthritis, multiple sclerosis, juvenile diabetes, asthma,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
lupus, diabetes, mellitus myasthenia gravis, psoriasis, dermatitis,
eczema, seborrhea, pulmonary inflammation, eye uveitis, hepatitis,
Grave's disease, Hashimoto's thyroiditis, Behcet's or Sjorgen's
syndrome, pernicious or immunohaemolytic anemia, idiopathic adrenal
insufficiency, polyglandular autoimmune syndrome,
glomerulonephritis, scleroderma, lichen planus, viteligo,
autoimmune thyroiditis, alveolitis, HTLV-1, HTLV-2, HIV-1, HIV-2,
nasopharyngeal carcinoma virus, HBV, HCV, HGV, yellow fever virus,
dengue fever virus, Japanese encephalitis virus, human papilloma
virus, Epstein-Barr, cytomegaloviruses, Herpes Simplex Type 1,
Herpes Simplex Type 2, Herpes Simplex Type 6, restenosis, stenosis,
artherosclerosis, lymphoma, leukemia, osteoarthritis, acute
pancreatitis, chronic pancreatitis, asthma, or adult respiratory
distress syndrome.
29-37. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. No. 61/162,013, filed Mar. 20,
2009; the contents of which are hereby incorporated by reference in
their entirety.
BACKGROUND OF THE INVENTION
[0003] Organisms must synthesize nucleotides in order for their
cells to divide and replicate. Nucleotide synthesis in mammals may
be achieved through one of two pathways: the de novo synthesis
pathway; or the salvage pathway. Different cell types use these
pathways to differing extents.
[0004] Inosine-5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205)
is an enzyme involved in the biosynthesis of guanine nucleotides.
IMPDH catalyzes the NAD-dependent oxidation of
inosine-5'-monophosphate (IMP) to xanthosine-5'-monophosphate (XMP)
[Jackson R. C. et. al., Nature, 256, pp. 331-333, (1975)].
Regardless of species, the reaction involves the random addition of
substrates. A conserved active site Cys residue attacks the C2
position of IMP and hydride is transferred to NAD, producing NADH
and the E-XMP* intermediate. NADH is released and a mobile flap
folds into the vacant NADH site, E-XMP* hydrolyzes and XMP is
released [W. Wang and L. Hedstrom, Biochemistry 36, pp. 8479-8483
(1997); J. Digits and L. Hedstrom, Biochemistry 38, pp. 2295-2306
(1999); Gan et al, Biochemistry 42, pp 847-863 (2003)]. The
hydrolysis step is at least partially rate-limiting in all of the
IMPDHs examined to date. The enzyme is unusual in that a large
conformational change occurs in the middle of a catalytic
cycle.
[0005] IMPDH is ubiquitous in eukaryotes, bacteria and protozoa [Y.
Natsumeda & S. F. Carr, Ann N.Y. Acad., 696, pp. 88-93 (1993)].
The prokaryotic forms share 30-40% sequence identity with the human
enzyme. Two isoforms of human IMPDH, designated type I and type II,
have been identified and sequenced [F. R. Collart and E. Huberman,
J. Biol. Chem., 263, pp. 15769-15772, (1988); Y. Natsumeda et. al.,
J. Biol. Chem., 265, pp. 5292-5295, (1990)]. Each is 514 amino
acids, and they share 84% sequence identity. Both IMPDH type I and
type II form active tetramers in solution, with subunit molecular
weights of 56 kDa [Y. Yamada et. al., Biochemistry, 27, pp.
2737-2745 (1988)].
[0006] The de novo synthesis of guanine nucleotides, and thus the
activity of IMPDH, is particularly important in B- and
T-lymphocytes. These cells depend on the de novo, rather than
salvage pathway to generate sufficient levels of nucleotides
necessary to initiate a proliferative response to mitogen or
antigen [A. C. Allison et. al., Lancet II, 1179, (1975) and A. C.
Allison et. al., Ciba Found. Symp., 48, 207, (1977)]. Thus, IMPDH
is an attractive target for selectively inhibiting the immune
system without also inhibiting the proliferation of other
cells.
[0007] Immunosuppression has been achieved by inhibiting a variety
of enzymes including, for example, the phosphatase calcineurin
(inhibited by cyclosporin and FK-506); dihydroorotate
dehydrogenase, an enzyme involved in the biosynthesis of
pyrimidines (inhibited by leflunomide and brequinar); the kinase
FRAP (inhibited by rapamycin); and the heat shock protein hsp70
(inhibited by deoxyspergualin). [See B. D. Kahan, Immunological
Reviews, 136, pp. 29-49 (1993); R. E. Morris, The Journal of Heart
and Lung Transplantation, 12(6), pp. S275-S286 (1993)].
[0008] Inhibitors of IMPDH are also known. U.S. Pat. Nos. 5,380,879
(incorporated by reference) and 5,444,072 (incorporated by
reference) and PCT publications WO 94/01105 and WO 94/12184
describe mycophenolic acid (MPA) and some of its derivatives as
potent, uncompetitive, reversible inhibitors of human IMPDH type I
(K.sub.i=33 nM) and type II (K.sub.i=9 nM). MPA has been
demonstrated to block the response of B- and T-cells to mitogen or
antigen [A. C. Allison et. al., Ann N.Y. Acad. Sci., 696, 63,
(1993)].
[0009] Immunosuppressants, such as MPA, are useful drugs in the
treatment of transplant rejection and autoimmune diseases. [R. E.
Morris, Kidney Intl., 49, Suppl. 53, S-26, (1996)]. However, MPA is
characterized by undesirable pharmacological properties, such as
gastrointestinal toxicity and poor bioavailability. [L. M. Shaw,
et. al., Therapeutic Drug Monitoring, 17, pp. 690-699, (1995)].
[0010] A novel noncompetitive inhibitor of meriniepodib, has
immunosuppressive activity, is orally bioavailable, and inhibits
the proliferation of primary human, mouse, rat, and dog lymphocytes
at concentrations of .about.100 M. Studies have demonstrated that
merimepodib is a potent, specific, and reversible IMPDH inhibitor
that selectively inhibits lymphocyte proliferation. It is currently
in clinical trials to treat hepatitis C virus.
[0011] Nucleoside analogs such as tiazofurin, ribavirin and
mizoribine also inhibit IMPDH [L. Hedstrom, et. al. Biochemistry,
29, pp. 849-854 (1990); L. Hedstrom, et al. Curr. Med. Chem. 1999,
6, 545-561]. These compounds require activation to either the
adenine dinucleotide (tiazofurin) or monophosphate derivatives
(ribavirin and mizoribine) that inhibit IMPDH. These activation
pathways are often absent in the cell of interest. In addition,
nucleoside analogs suffer from lack of selectivity and can be
further metabolized to produce inhibitors of other enzymes.
Therefore, nucleoside analogs are prone to toxic side effects.
[0012] Mycophenolate mofetil, a prodrug which quickly liberates
free MPA in vivo, was recently approved to prevent acute renal
allograft rejection following kidney transplantation. [L. M. Shaw,
et. al., Therapeutic Drug Monitoring, 17, pp. 690-699, (1995); H.
W. Sollinger, Transplantation, 60, pp. 225-232 (1995)]. Several
clinical observations, however, limit the therapeutic potential of
this drug. [L. M. Shaw, et. al., Therapeutic Drug Monitoring, 17,
pp. 690-699, (1995)]. MPA is rapidly metabolized to the inactive
glucuronide in vivo. [A. C., Allison and E. M. Eugui, Immunological
Reviews, 136, pp. 5-28 (1993)]. The glucuronide then undergoes
enterohepatic recycling causing accumulation of MPA in the
gastrointestinal tract where it cannot exert its IMPDH inhibitory
activity on the immune system. This fact effectively lowers the
drug's in vivo potency, while increasing its undesirable
gastrointestinal side effects.
[0013] IMPDH also plays a role in other physiological events.
Increased IMPDH activity has been observed in rapidly proliferating
human leukemic cell lines and other tumor cell lines, indicating
IMPDH as a target for anti-cancer as well as immunosuppressive
chemotherapy [M. Nagai et. al., Cancer Res., 51, pp. 3886-3890,
(1991)]. IMPDH has also been shown to play a role in the
proliferation of smooth muscle cells, indicating that inhibitors of
IMPDH, such as MPA, may be useful in preventing restenosis or other
hyperproliferative vascular diseases [C. R. Gregory et al.,
Transplantation, 59, pp. 655-61 (1995); PCT publication WO
94/12184; and PCT publication WO 94/01105].
[0014] Additionally, IMPDH has been shown to play a role in viral
replication in some viral cell lines. [S. F. Carr, J. Biol. Chem.,
268, pp. 27286-27290 (1993)]. Analogous to lymphocyte and tumor
cell lines, the implication is that the de novo, rather than the
salvage, pathway is critical in the process of viral
replication.
[0015] Cryptosporidiosis is a severe gastrointestinal disease
caused by protozoan parasites of the genus Cryptosporidium. The
most common causes of human disease are C. parvum and C. hominis,
though disease can also result from C. felis, C. meleagridis, C.
canis, and C. muris infection. Small children, pregnant women, the
elderly, and immuno-compromised people (e.g., AIDS patients) are at
risk of severe, chronic and often fatal infection. [Carey, C. M.,
Lee, H., and Trevors, J. T., Water Res., 38, 818-62 (2004); and
Fayer, R., Veterinary Parasitology, 126, 37-56 (2004)]. The
Cryptosporidium parasites produce spore-like oocysts that are
highly resistant to water chlorination. Several large outbreaks in
the U.S. have been linked to drinking and recreational water.
Infection rates are extremely high, with disease manifest in 30% of
exposed individuals and a 50-70% mortality rate among
immuno-compromised individuals. Furthermore, there is a growing and
credible concern that these organisms could be deliberately
introduced into the water supply in an act of bioterrorism.
Effective drugs are urgently needed for the management of
cryptosporidiosis in AIDS patients and/or epidemic outbreaks.
[0016] All parasitic protozoa lack purine biosynthetic enzymes and
must salvage purines from their hosts, making this pathway an
extremely attractive target for developing anti-protozoal drugs.
IMPDH is a key enzyme in the purine salvage pathway of C. parvum.
As discussed above, IMPDH is a validated drug target in
immunosuppressive, cancer and viral therapy, so the human enzymes
are extremely well studied. It has recently been shown that C.
parvum IMPDH has very different properties than the human enzymes
and that IMPDH inhibitors block parasite proliferation in vivo [N.
N. Umejiego et al, J Biol Chem, 279 pp. 40320-40327 (2004); and B.
Striepen et al, Proc Natl Acad Sci USA, 101 pp. 3154-9 (2004)].
[0017] Thus, there exists a need for potent IMPDH inhibitors with
improved pharmacological properties and selectivities. Such
inhibitors should have therapeutic potential as immunosuppressants,
anti-cancer agents, anti-vascular hyperproliferative agents,
antiinflammatory agents, antifungal agents, antipsoriatic and
anti-viral agents. Specifically, there is a need for selective
IMPDH inhibitors that can slow or block parasite and bacterial
proliferation. The present invention fulfills this need and has
other related advantages.
SUMMARY OF THE INVENTION
[0018] One aspect of the present invention relates to compounds,
and pharmaceutically acceptable salts and prodrugs thereof, which
are useful as inhibitors of IMPDH. In certain embodiments, a
compound of the invention selectively inhibits a parasitic IMPDH
versus a host (e.g., mammalian) IMPDH. Further, the invention
provides pharmaceutical compositions comprising one or more
compounds of the invention. The invention also relates to methods
of treating various parasitic and bacterial infections in mammals.
Moreover, the compounds may be used alone or in combination with
other therapeutic or prophylactic agents, such as anti-virals,
anti-inflammatory agents, antimicrobials and
immunosuppressants.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 depicts triazole compounds 1-7 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0020] FIG. 2 depicts triazole compounds 8-14 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0021] FIG. 3 depicts triazole compounds 15-21 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0022] FIG. 4 depicts triazole compounds 22-28 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0023] FIG. 5 depicts triazole compounds 29-34 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0024] FIG. 6 depicts triazole compounds 35-39 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0025] FIG. 7 depicts a general scheme for the preparation of
various 1,2,3-triazoles. Reagents and conditions: X and Y.dbd.N,
CH, or CCl. (a) [R.sup.1=Me]MeCH(OH)C.ident.CH, 0.degree. C.,
Ph.sub.3P, 10 min, DEAD, rt, 12 h; (b)
[R.sup.1.dbd.H]BrCHC.ident.CH, K.sub.2CO.sub.3, DMF, rt, 12 h; (c)
[R.sup.1=i-Pr, R.sup.2.dbd.CO.sub.2H] (i) LiAlH.sub.4, THF,
0.degree. C., 4 h, (ii) (COCl).sub.2, DMSO, DCM, Et.sub.3N,
-78.degree. C., 3 h; (d) [R.sup.1=Et, R.sup.2.dbd.CO.sub.2Et]
DIBAL, THF, -78.degree. C., 6 h; (e) (i) CBr.sub.4, Ph.sub.3P, DCM,
0.degree. C., 2 h, (ii) n-BuLi, THF, -78.degree. C., 2 h; (f)
R.sup.3PhN.sub.3, CH.sub.3CN, DIPA, CuI, rt, 30 min; (g) m-CPBA,
DCM, 0.degree. C., 12 h.
[0026] FIG. 8 depicts the synthesis of 41 [R.sup.1Et,
R.sup.2.dbd.CO.sub.2Et]. Reagents and conditions: (a) (i) c-PrMgBr,
THF, -20.degree. C., 2 h, (ii) Ph.sub.3P, CBr.sub.4, DCM, 0.degree.
C., 2 h; (b) 1-naphthol, K.sub.2CO.sub.3, DMF, rt, 2 h; (c) 3 M
NaOH, THF:H.sub.2O (2:1), 80.degree. C., 6 h.
[0027] FIG. 9 depicts a representative synthetic scheme for the
formation of triazoles 51.
[0028] FIG. 10 depicts the IC.sub.50 determinations for inhibition
of C. parvum IMPDH by 1,2,3-triazole derivatives; .sup.a=0.05%
Fatty acid free bovine serum albumin; .sup.b=Not Determined.
[0029] FIG. 11 depicts oxadiazole compound 52 and its IC.sub.50
value against recombinant C. parvum IMPDH.
[0030] FIG. 12 depicts amide compounds 53-57 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0031] FIG. 13 depicts amide compounds 58-67 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0032] FIG. 14 depicts amide compounds 68-76 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0033] FIG. 15 depicts amide and ester compounds 77-85 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0034] FIG. 16 depicts amide compounds 86-95 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0035] FIG. 17 depicts amide compounds 96-104 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0036] FIG. 18 depicts amide compounds 105-113 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0037] FIG. 19 depicts amide and ketone compounds 114-120 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0038] FIG. 20 depicts amide compounds 121-122 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0039] FIG. 21 depicts amide compounds 123-124 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0040] FIG. 22 depicts amide compounds 125-129 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0041] FIG. 23 depicts amide compounds 130-134 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0042] FIG. 24 depicts compounds 135-140 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0043] FIG. 25 depicts amide compounds 141-145 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0044] FIG. 26 depicts compounds 146-148 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0045] FIG. 27 depicts amide compounds 149-152 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0046] FIG. 28 depicts amide compounds 153-156 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0047] FIG. 29 depicts amide compounds 157-160 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0048] FIG. 30 depicts amide compounds 161-162 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0049] FIG. 31 depicts amide compounds 163-167 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0050] FIG. 32 depicts amide compounds 168-172 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0051] FIG. 33 depicts amide compounds 173-177 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0052] FIG. 34 depicts amide, ester, and ketone compounds 178-185
and their respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0053] FIG. 35 depicts two syntheses of 188. Reagents and
conditions: (a) (i) c-PrMgBr, THF, -20.degree. C., 2 h, (ii)
Ph.sub.3P, CBr.sub.4, DCM, 0.degree. C., 2 h; (b) 1-naphthol,
K.sub.2CO.sub.3, DMF, rt, 2 h; (c) 3 M NaOH, THF:H.sub.2O (2:1),
80.degree. C., 6 h; (d) 4-chloroaniline, 0.degree. C., EDCI.HCl,
rt, 12 h; (e) 4-chloroaniline, cat. DMAP, DCM, rt, 2 h; (f)
4-hydroxyquinoline, K.sub.2CO.sub.3, DMF, 0.degree. C., rt, 12
h.
[0054] FIG. 36 depicts IC.sub.50 values for inhibition of
recombinant C. parvum IMPDH by amide derivatives; .sup.a=0.05%
Fatty acid free bovine serum albumin; .sup.b=Not Determined.
[0055] FIG. 37 depicts triazole compounds A111-A113 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0056] FIG. 38 depicts triazole compounds A114-A119 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0057] FIG. 39 depicts amide compounds C68-C70 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0058] FIG. 40 depicts amide compounds C71-C85 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0059] FIG. 41 depicts amide compounds C86-C100 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0060] FIG. 42 depicts phthalazinone compounds D1-D18 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0061] FIG. 43 depicts phthalazinone compounds D19-D36 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0062] FIG. 44 depicts phthalazinone compounds D37-D54 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0063] FIG. 45 depicts phthalazinone compounds D55-D61 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0064] FIG. 46 depicts pyrazole compounds N1-N18 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0065] FIG. 47 depicts pyrazole compounds N19-N26 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0066] FIG. 48 depicts urea compounds P1-P15 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0067] FIG. 49 depicts urea compounds P16-P32 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0068] FIG. 50 depicts urea compounds P33-P51 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0069] FIG. 51 depicts urea compounds P52-P68 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0070] FIG. 52 depicts urea compounds P69-P80 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0071] FIG. 53 depicts urea compounds P81-P97 and their respective
IC.sub.50 values against recombinant C. parvum IMPDH.
[0072] FIG. 54 depicts benzoxazole compounds Q1-Q15 and their
respective IC.sub.50 values against recombinant C. parvum
IMPDH.
[0073] FIG. 55 tabulates inhibition of recombinant C. parvum IMPDH
(CpIMPDH) and human IMPDH type 2 (hIMPDH2). b. .ltoreq.20%
inhibition at 50 .mu.M; c. .ltoreq.20% inhibition at 5 .mu.M; d.
.ltoreq.10% inhibition at 5 .mu.M; e. .ltoreq.20% inhibition
observed at 2 .mu.M.
[0074] FIG. 56 tabulates the results of metabolic and plasma
stability studies on various compounds of the invention.
[0075] FIG. 57 depicts validation of the T. gondii-CpIMPDH reporter
parasite. Schematics of the routes to GMP for the wild-type T.
gondii, T. gondii-.DELTA.HXGPRT, and T. gondii-CpIMPDH are shown in
A, D & G respectively. Genetic studies have shown that the
salvage of adenosine via adenosine kinase is the predominant route
to GMP for T. gondii and IMPDH catalyzes the rate limiting step of
this pathway. However, in the absence of adenosine kinase, TgHXGPRT
allows for the salvage of adenosine, adenine and guanosine such
that the activity of TgHXGPRT is sufficient for parasite
proliferation. Several transporters for the uptake of nucleobases
and nucleotides have been characterized in T. gondii. Unlike T.
gondii and other Apicomplexa, C. parvum lacks HXGPRT and is
dependent on the salvage of adenosine and thus the activity of
CpIMPDH. A single adenosine transporter has been identified in the
genome of C. parvum. The T. gondii pathways shown in grey highlight
the genes disrupted in the parasite clones used in this study,
TgHXGRT in a previous study (HXGPRT) and TgIMPDH in this study.
Hyp, hypoxanthine; Xan, xanthine; Gua, guanine; Guo, guanosine;
Ade, adenine; Ado, adenosine; Ino, inosine; AMP, adenosine
monophosphate; IMP, inosine monophosphate; XMP, xanthosine
monophosphate; GMP, guanosine monophosphate; HXGPRT, hypoxanthine
xanthine gunanine phosphoribosyltransferase; IMPDH, IMP
dehydrogenase, 1, adenine deaminase; 2, adenosine deaminase; 3,
purine nucleoside phosphorylase; 4, adenosine kinase; 5, AMP
deaminase; 6, adenoylsuccinate synthase and adenoylsuccinate lyase;
7, GMP synthase. Panels B, E & H show parasite growth in the
presence of 0 .mu.M and 7.8 .mu.M MPA for wild-type T. gondii, T.
gondii-AHXGPRT, and T. gondii-CpIMPDH respectively. Panels C, F,
and I show parasite growth curves in the presence of 0 .mu.M and
7.8 .mu.M MPA, with the addition of 0.33 mM xanthine to the culture
media, for wild-type T. gondii, T. gondii-.DELTA.HXGPRT, and T.
gondii-CpIMPDH respectively. Data are representative of two
independent experiments.
[0076] FIG. 58 depicts an overview and validation of the high
content imaging C. parvum growth assay. A, schematic representation
of differential labelling of parasite and host. B, detail of an
exemplary micrograph obtained through the screening routine.
Numbers indicate object identifies after segmentation analysis.
Panel C shows a 2-fold titration of C. parvum oocysts where the top
concentration was 1.2.times.10.sup.6 oocysts per well. For panel D,
the ratio of the number of FITC-VVL labelled C. parvum parasites to
DAPI labelled HCT-8 host cell nuclei was used to standardize each
well and percent C. parvum growth (solid line) was normalized to
parasites receiving DMSO alone. The paromomycin EC.sub.50 for C.
parvum was growth was 97 .mu.M. Paromomycin in addition to reducing
parasite number also reduces the average size of the parasite
(dashed line). The mean parasite area was measured per well for
each treatment in triplicate. The percent area was then calculated
by normalizing to the mean area of parasites receiving DMSO alone.
Data shows the mean of two independent experiments set up with
triplicate wells in a 96-well format.
[0077] FIG. 59 depicts the identification of derivatives with high
potency and selectivity in the T. gondii-CpIMPDH model. Panel A
shows the EC.sub.50 for a selection of compounds assayed in the T.
gondii-CpIMPDH parasite model and demonstrates a range in compound
selectivity and potency. Compounds were assayed in triplicate and
growth inhibition was calculated on a day during the exponential
phase of growth, by normalization to wells receiving DMSO alone.
The EC.sub.50 calculation was performed as described in FIG. 63.
Compounds A82, A89, A90, A92, A102, A103, A105, and A110 were
selected for rescreening and the mean for at least 2 replicate
experiments are shown. These compounds were then tested for
inhibition of C. parvum (panel B) and host cell growth (panel C).
For panel B, percent C. parvum growth was determined using the
high-content imaging assay, with compound at 12.5 .mu.M and 25
.mu.M. The ratio of FITC-VVL labeled C. parvum parasites to DAPI
labeled HCT-8 host cell nuclei was used to standardize each well
and percent growth was normalized to parasites receiving DMSO
alone, the mean of triplicate wells is shown. A selection of
compounds were selected for re-screening and the mean over at least
2 replicate experiments is shown for compounds A90, A92, A98, A103,
A105, A109 and A110. Panel C shows percent host cell growth assayed
using the pmaxGFP fluorescent HCT-8 cell line with compound at 12.5
.mu.M and 25 .mu.M. GFP expressing HCT-8 cells were seeded at 4000
cells per well into 96-well plates and triplicate wells were spiked
with test compound. Fluorescence was measured daily with a
SpectraMax M22/M2e (Molecular Devices) plate reader (Ex 485, Em
530) for 7 days. Percent growth inhibition was calculated on a day
during the exponential phase of growth, by normalization to wells
receiving DMSO alone. A selection of compounds A89, A90 and A92
were selected for re-screening and the mean over at least two
replicate experiments is shown.
[0078] FIG. 60 depicts the correlation between CpIMPDH enzyme
inhibition and potency and selectivity in the T. gondii-CpIMPDH
model. Panel Ashows a relatively weak (r=0.58) and statistically
insignificant (p-value=0.3) correlation between the compound
IC.sub.50 values for the CpIMPDH enzyme and the EC.sub.50 for
proliferation of the T. gondii-CpIMPDH parasite. However a strong,
positive correlation exists between the potency of CpIMPDH enzyme
inhibition when assayed in the presence of BSA and inhibition of T.
gondii-CpIMPDH proliferation (r=-0.94, p<0.0001; panel B). Panel
C, shows that selectivity in the T. gondii model, determined by the
relative inhibition of the T. gondii-CpIMPDH parasite over
wild-type T. gondii clone, also correlates well with the potency of
enzyme inhibition in the presence of BSA (r=-0.92,
p<0.0001).
[0079] FIG. 61 depicts that compounds A103 and A110 are potent
inhibitors of C. parvum growth. C. parvum growth was determined
using the HCl assay. The ratio of the number of FITC-VVL labelled
C. parvum parasites to DAPI labelled HCT-8 host cell nuclei was
used to standardize each well and percent C. parvum growth was
normalised to parasites receiving DMSO alone. Panels A and B show
compounds A103 and A110 respectively (EC.sub.50<0.8 .mu.M). Data
shows the mean of two independent experiments with triplicate
wells.
[0080] FIG. 62 tabulates data for various compounds in enzyme
assays (in the absence and presence of BSA), surrogate T. gondii
model assay, host cell growth and tissue culture model of C. parvum
infection. N.A., not applicable; N.D., not determined. a.
Selectivity=T. gondii-CpIMPDH EC.sub.50 versus wild-type T. gondii
EC.sub.50; b. highest concentration tested; c. Maurya et al; d.
lowest concentration tested; e. Umejiego et al.; f. qPCR assay.
[0081] FIG. 63 depicts an overview of obtaining an EC.sub.50 for T.
gondii growth. Fluorescent T. gondii parasites are seeded into
96-well plates and spiked with test compound. Fluorescence is
measured daily with a SpectraMax M22/M2e (Molecular Devices) plate
reader for 6-7 days. The fluorescence readings on a day during the
exponential phase of the growth curve, for example day 4 in panel
A, are used to calculated percent growth inhibition. These values
are fitted to using the 4 parameter model y=D+(A-D)/(1+(x/C).sup.B)
where D is the minimum value, A is the maximum value, C is the EC50
and B is the Hill coefficient, using the SoftMax Pro v5 software,
as illustrated in panel B. The absolute EC.sub.50 is recorded at
the x intercept where y=50.
[0082] FIG. 64 shows results of various compounds in the surrogate
T. gondii model. Panel A shows the EC.sub.50 for a selection of
compounds assayed in the T. gondii-CpIMPDH parasite model.
Compounds were assayed in triplicate and growth inhibition was
calculated on a day during the exponential phase of growth, by
normalization to wells receiving DMSO alone. The EC.sub.50
calculation was performed as described in figure S2. Note the
highest concentration tested in panel A was for compound A30 was 20
.mu.M. Panel B shows percent host cell growth assayed using the
pmaxGFP fluorescent HCT-8 cell line with compound at 25 .mu.M and
50 .mu.M. GFP expressing HCT-8 cells were seeded at 4000 cells per
well into 96-well plates and triplicate wells were spiked with test
compound. Fluorescence was measured daily with a SpectraMax M22/M2e
(Molecular Devices) plate reader (Ex 485, Em 530) for 7 days.
Percent growth inhibition was calculated on a day during the
exponential phase of growth, by normalization to wells receiving
DMSO alone.
[0083] FIG. 65 depicts the selectivity of various compounds in the
surrogate Toxo/CpIMPDH assay.
[0084] FIG. 66 tabulates activity levels of various compounds; the
surrogate Toxoplasma model is predictive for anti-Cryptosporidium
activity.
[0085] FIG. 67 tabulates the inhibition of various IMPDHs by
compounds A-H. Cp, C. parvum; Hp, Helicobacter pylori; Bb, Borrelia
burgdorferi; Sp, Streptococcus pylori; ECIMPDH S250A/L444Y,
Escherichia coli IMPDH containing an alanine residue at serine-240
and a leucine residue at tyrosine-444. These compounds (100 .mu.M)
do not inhibit IMPDHs from E. coli, Leishmania donovanii and
Tritrichomonas foetus. "Intrinsic" values (adjusted for the
competition with the mobile flap) are shown in parentheses.
[0086] FIG. 68 depicts the IMPDH reaction: a. Chemical mechanism: a
conserved Cys attacks C2 of IMP and hydride is transferred to
NAD.sup.+ producing the covalent intermediate E-XMP*. E-XMP* is
hydrolyzed with a conserved Arg residue acting as a general base to
produce XMP. b. The hydride transfer reaction proceeds in an open
enzyme conformation. After NADH departs, a mobile flap folds into
the NAD site, carrying the catalytic Arg into the active site
Inhibitors compete with the flap, so the equilibrium between open
and closed states is a determinant of inhibitor affinity. c.
Phylogenetic tree of IMPDHs.
[0087] FIG. 69 depicts that C91 inhibits H. pylori growth. CFU,
colony forming units. Filled circles, DMSO alone. C91
concentrations: open circles, 2 .mu.M; closed squares, 7 .mu.M;
open squares, 20 .mu.M; closed triangles, 60 .mu.M; open triangles,
200 .mu.M.
[0088] FIG. 70 depicts the x-ray crystal structure of CpIMPDH with
IMP and C64 shown from two different perspectives. The electron
density map prior to C64 modeling with coefficients 2Fo-Fc is
contoured to 1.sigma. and shown as a slate cage. The electron
density map prior to C64 modeling with coefficients Fo-Fc is
contoured to 3.sigma.. Bromine K-edge peak anomalous dispersion map
is contoured to 4.sigma..
[0089] FIG. 71 depicts the C64 binding pocket of CpIMPDH superposed
with human IMPDH2. CpIMPDH residues are labeled.
DETAILED DESCRIPTION
Overview
[0090] One aspect of the present invention relates to compounds,
and pharmaceutically acceptable salts and prodrugs thereof, which
are useful as inhibitors of IMPDH. In certain embodiments, a
compound of the invention selectively inhibits a parasitic or
bacterial IMPDH versus a host (e.g., mammalian) IMPDH. In certain
embodiments, the present invention relates to selective inhibition
of Cryptosporidium IMPDH in the presence of human
inosine-5'-monophosphate-dehydrogenase (IMPDH type I and type II).
Further, the invention provides pharmaceutical compositions
comprising one or more compounds of the invention. The invention
also relates to methods of treating various parasitic and bacterial
infections in mammals. Moreover, the compounds may be used alone or
in combination with other therapeutic or prophylactic agents, such
as anti-virals, anti-inflammatory agents, antimicrobials and
immunosuppressants.
[0091] IMPDH-Mediated Diseases. IMPDH-mediated disease refers to
any disease state in which the IMPDH enzyme plays a regulatory role
in the metabolic pathway of that disease. Examples of
IMPDH-mediated disease include transplant rejection and autoimmune
diseases, such as rheumatoid arthritis, multiple sclerosis,
juvenile diabetes, asthma, and inflammatory bowel disease, as well
as other inflammatory diseases, cancer, viral replication diseases
and vascular diseases.
[0092] For example, the compounds, compositions and methods of
using them of the invention may be used in the treatment of
transplant rejection (e.g., kidney, liver, heart, lung, pancreas
(islet cells), bone marrow, cornea, small bowel and skin allografts
and heart valve xenografts) and autoimmune diseases, such as
rheumatoid arthritis, multiple sclerosis, juvenile diabetes,
asthma, inflammatory bowel disease (Crohn's disease, ulcerative
colitus), lupus, diabetes, mellitus myasthenia gravis, psoriasis,
dermatitis, eczema, seborrhea, pulmonary inflammation, eye uveitis,
hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet's or
Sjorgen's syndrome (dry eyes/mouth), pernicious or immunohaemolytic
anemia, idiopathic adrenal insufficiency, polyglandular autoimmune
syndrome, and glomerulonephritis, scleroderma, lichen planus,
viteligo (depigmentation of the skin), autoimmune thyroiditis, and
alveolitis, inflammatory diseases such as osteoarthritis, acute
pancreatitis, chronic pancreatitis, asthma and adult respiratory
distress syndrome, as well as in the treatment of cancer and
tumors, such as solid tumors, lymphomas and leukemia, vascular
diseases, such as restenosis, stenosis and artherosclerosis, and
DNA and RNA viral replication diseases, such as retroviral
diseases, and herpes.
[0093] Selective Inhibition of Microbial IMPDH. IMPDH enzymes are
also known to be present in bacteria, fungi, and protozoans and
thus may regulate microbial growth. As such, the IMPDH-inhibitor
compounds, compositions and methods described herein may be useful
as antibacterials, antifungals, and/or antiprotozoans, either alone
or in combination with other anti-microbial agents.
[0094] Microbial inhibition can be measured by various methods,
including, for example, IMPDH HPLC assays (measuring enzymatic
production of XMP and NADH from IMP and NAD), IMPDH
spectrophotometric assays (measuring enzymatic production of NADH
from NAD or XMP from IMP), IMPDH fluorometric assays (measuring
enzymatic production of NADH from NAD), IMPDH radioassays
(measuring enzymatic production of radiolabeled XMP from
radiolabeled IMP or tritium release into water from 2.sup.-3H-IMP).
[See C. Montero et al., Clinica Chimica Acta, 238, pp. 169-178
(1995)]. Additional assays known in the art can be used in
ascertaining the degree of activity of an inventive compound as an
IMPDH inhibitor. For example, activity of IMPDH I and IMPDH II can
be measured following an adaptation of the method described in WO
97/40028. [See, additionally, U.S. Patent Application 2004/0102497
(incorporated by reference)].
[0095] Accordingly, in certain embodiments, the inventive compounds
are capable of targeting and selectively inhibiting the IMPDH
enzyme in bacteria. It is known that knocking out the IMPDH gene
makes some bacteria avirulent, while has no effect on others. The
effectiveness probably depends on which salvage pathways are
operational in a given bacteria, and the environmental niche of the
infection. It has been shown that IMPDHs from H. pylori, S.
pyogenes and B. burgdorferi are sensitive to the inhibitors of the
invention, and that the growth of H. pylori is blocked by
inhibitors of the invention. It is also expected that various
Campylobacter, Arcobacter, Bacteroides, Fusobacterium,
Burkholderia, Clostridia, Neisseria, Mycobacterium, or
Acinetobacter organisms will be inhibited by the compounds
described herein. Organisms belonging to these genera are
responsible for illnesses such as ulcers and acid reflux (H.
pylori), Lyme disease (B. burgdorferi), infection (S. pyogenes),
food poisoning (C. jejuni and A. butzleri), abscesses (B.
capillosis), periodontitis (F. nucleatum), skin ulcers (F.
nucleatum), Lemierre's syndrome (F. nucleatum), infection in cystic
fibrosis (B. cenocepacia), pneumonia (S. pneumoniae), botulism (C.
botulinum), gonorrhea (N. gonorrhoeae), tuberculosis (M.
tuberculosis), leprosy (M. leprae), and drug resistant infection
(A. baumannii). In addition, Staphylococcus and Bacillus anthracis
are sensitive to mycophenolic acid, suggesting that IMPDH
inhibitors of the invention may also be effective against these
bacteria.
[0096] In addition, in certain embodiments, these compounds are
capable of targeting and selectively inhibiting the IMPDH enzyme in
fungi, as evidenced by the mycophenolic acid sensitivity of
Saccharomyces cerevisiae, Candida albicans, Cryptococcus
neoformans, Aspergillus flavus and Trichophyton.
[0097] Further, in certain embodiments, the inventive compounds are
capable of targeting and selectively inhibiting the IMPDH enzyme in
protozoans, such as Toxoplasma, Eimeria, Cryptosporidium,
Plasmodium, Babesia, Theileria, Neospora, Sarcocystis, Giardia,
Entamoeba, Trichomonas, Leishmania and Trypanosoma. In certain
embodiments, these compounds are capable of targeting and
selectively inhibiting the IMPDH enzyme in Cryptosporidium parvum
and other Cryptosporidium species.
Selected Compounds of the Invention.
[0098] Triazole Series
[0099] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula I:
##STR00001##
[0100] wherein, independently for each occurrence,
[0101] R.sup.1 is hydrogen, alkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl;
[0102] R.sup.2 is hydrogen or alkyl; [0103] or R.sup.1 and an
instance of R.sup.2 taken together with the carbon atoms to which
they are attached form a 5-, 6-, or 7-membered aryl or heteroaryl
ring;
[0104] R.sup.3 is hydrogen or alkyl;
[0105] Y.sup.1 is absent, O, or NR.sup.4;
[0106] Y.sup.2 is absent, O, NR.sup.4, alkylene,
--(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--,
--(CH.sub.2).sub.m--NR.sup.4--(CH.sub.2).sub.p--,
--(CH.sub.2).sub.m--C(.dbd.O)--(CH.sub.2).sub.p--,
--(CH.sub.2).sub.mC(.dbd.O)NR.sup.4--(CH.sub.2).sub.p--, or
--(CH.sub.2).sub.mC(.dbd.O)O--(CH.sub.2).sub.p--;
[0107] n is 0, 1, 2, 3, or 4;
##STR00002##
is aryl or heteroaryl;
##STR00003##
is hydrogen, aryl, or heteroaryl;
[0108] R.sup.4 is hydrogen or alkyl;
[0109] m is 0, 1, 2, 3, or 4; and
[0110] p is 0, 1, or 2; [0111] wherein, any of the aforementioned
alkyl, aryl, heteroaryl, or aralkyl may be substituted with one or
more groups independently selected from the group consisting of
halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy,
alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,
acyloxy, silyl, alkylthio, sulfonate, sulfonyl, sulfonamido,
formyl, cyano, and isocyano.
[0112] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Y.sup.1 is O or absent.
[0113] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Y.sup.1 is O.
[0114] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Y.sup.1 is absent.
[0115] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is aryl or
hydrogen.
[0116] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is aryl.
[0117] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is phenyl.
[0118] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is hydrogen.
[0119] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 1, 2, 3, or 4.
[0120] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 0 or 1.
[0121] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 1.
[0122] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 0.
[0123] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is hydrogen or
alkyl.
[0124] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is hydrogen.
[0125] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is alkyl.
[0126] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is methyl, ethyl,
n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl.
[0127] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is methyl.
[0128] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is ethyl.
[0129] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is i-propyl.
[0130] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 and an instance of
R.sup.2 taken together with the carbon atoms to which they are
attached form a 5- or 6-membered aryl ring.
[0131] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 and an instance of
R.sup.2 taken together with the carbon atoms to which they are
attached form a 6-membered aryl ring.
[0132] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.3 is hydrogen or
alkyl.
[0133] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.3 is hydrogen.
[0134] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Y.sup.2 is absent,
--(CH.sub.2).sub.mC(.dbd.O)NR.sup.4--(CH.sub.2).sub.p--, or
--(CH.sub.2).sub.mC(.dbd.O)O--(CH.sub.2).sub.p--.
[0135] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Y.sup.2 is absent.
[0136] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Y.sup.2 is
--(CH.sub.2).sub.mC(.dbd.O)NR.sup.4--(CH.sub.2).sub.p--.
[0137] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Y.sup.2 is
--(CH.sub.2).sub.mC(.dbd.O)NR.sup.4--(CH.sub.2).sub.p--; R.sup.4 is
hydrogen; m is 1; and p is 0.
[0138] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00004##
R.sup.5 is halo, azido, alkyl, haloalkyl, aralkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,
hydroxy, alkoxy, haloalkyloxy, aryloxy, heteroaryloxy, amino,
nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,
carboxyl, alkoxycarbonyl, acyloxy, alkylthio, sulfonate, sulfonyl,
sulfonamido, formyl, cyano, or isocyano; and q is 0 to 5
inclusive.
[0139] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00005##
and R.sup.5 is selected from the group consisting of halo, alkoxy,
haloalkyloxy, alkylthio, amido, and cyano.
[0140] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00006##
and R.sup.5 is halo.
[0141] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00007##
and R.sup.5 is chloro.
[0142] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00008##
and R.sup.5 is bromo.
[0143] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00009##
and R.sup.5 is alkoxy.
[0144] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00010##
and R.sup.5 is methoxy.
[0145] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00011##
and R.sup.5 is haloalkyloxy.
[0146] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00012##
and R.sup.5 is trifluoromethoxy.
[0147] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00013##
and R.sup.5 is alkylthio.
[0148] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00014##
and R.sup.5 is methylthio.
[0149] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00015##
and R.sup.5 is cyano.
[0150] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00016##
and R.sup.5 is halo.
[0151] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00017##
and R.sup.5 is chloro.
[0152] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00018##
and R.sup.5 is halo or cyano.
[0153] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00019##
and R.sup.5 is halo.
[0154] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00020##
and R.sup.5 is chloro.
[0155] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00021##
one instance of R.sup.5 is halo; and one instance of R.sup.5 is
cyano.
[0156] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00022##
one instance of R.sup.5 is halo; and one instance of R.sup.5 is
amido.
[0157] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00023##
one instance of R.sup.5 is chloro; and one instance of R.sup.5 is
cyano.
[0158] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00024##
and R.sup.5 is halo.
[0159] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00025##
and R.sup.5 is chloro.
[0160] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00026##
[0161] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00027##
[0162] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00028##
is hydrogen or selected from the group consisting of
##STR00029##
q is 0 to 5, inclusive; Z is --N-- or --CH--;
##STR00030##
is selected from the group consisting of hydrogen, alkyl, aryl, and
heteroaryl; and R.sup.5 is halo, azido, alkyl, haloalkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
heteroaralkyl, hydroxy, alkoxy, haloalkyloxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, alkylthio,
sulfonate, sulfonyl, sulfonamido, formyl, cyano, or isocyano; and
R.sup.6 is hydrogen or alkyl.
[0163] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00031##
[0164] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00032##
and R.sup.5 is halo.
[0165] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00033##
and R.sup.5 is chloro.
[0166] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00034##
and R.sup.5 is halo.
[0167] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00035##
and R.sup.5 is chloro.
[0168] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00036##
[0169] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00037##
[0170] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00038##
[0171] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00039##
[0172] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00040##
[0173] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00041##
[0174] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00042##
[0175] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00043##
is hydrogen.
[0176] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00044##
[0177] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00045##
[0178] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051##
[0179] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00052## ##STR00053##
[0180] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula II:
##STR00054##
[0181] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula III:
##STR00055##
[0182] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula IV:
##STR00056##
[0183] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula V:
##STR00057##
[0184] Amide Series
[0185] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula VI:
##STR00058##
[0186] wherein, independently for each occurrence, [0187] R.sup.1
is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl; [0188] R.sup.2 is hydrogen or alkyl; [0189] R.sup.3
is hydrogen or alkyl; [0190] n is 0, 1, 2, 3, or 4; [0191] X is
absent, alkylene, --NR.sup.3--, --SO.sub.2--, or
--CR.sup.3.dbd.N--; [0192] Z is --N.dbd. or --CR.sup.5.dbd.;
[0192] ##STR00059## [0193] is aryl or heteroaryl;
[0193] ##STR00060## [0194] is selected from the group consisting of
hydrogen, alkyl, aryl, and heteroaryl; [0195] q is 0, 1, 2, 3, or
4; and [0196] R.sup.5 is halo, azido, alkyl, haloalkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
heteroaralkyl, hydroxy, alkoxy, haloalkyloxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, alkylthio,
sulfonate, sulfonyl, sulfonamido, formyl, cyano, or isocyano;
[0197] wherein, any of the aforementioned alkyl, aryl, heteroaryl,
or aralkyl may be substituted with one or more groups independently
selected from the group consisting of halo, azido, alkyl,
haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, silyl,
alkylthio, sulfonate, sulfonyl, sulfonamido, formyl, cyano, and
isocyano.
[0198] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is absent, methylene,
--NH--, --SO.sub.2--, or --CH.dbd.N--.
[0199] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is absent.
[0200] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is methylene.
[0201] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is --NH--.
[0202] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is --SO.sub.2--.
[0203] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is --CH.dbd.N--.
[0204] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein q is 0.
[0205] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein q is 1.
[0206] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein q is 1; and R.sup.5 is
halo.
[0207] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein q is 1; and R.sup.5 is
bromo.
[0208] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein Z is --N.dbd..
[0209] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 1, 2, 3, or 4.
[0210] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 1 or 2.
[0211] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 1.
[0212] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein n is 2.
[0213] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is hydrogen or
alkyl.
[0214] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is hydrogen.
[0215] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is alkyl.
[0216] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.1 is methyl, ethyl,
n-propyl, or i-propyl.
[0217] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is hydrogen.
[0218] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.3 is hydrogen.
[0219] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00061##
and p is 0, 1, 2, or 3.
[0220] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00062##
and R.sup.5 is amido, alkoxy, halo, haloalkyl, aryl, haloaryl,
alkyl, hydroxy, alkylthio, sulfonyl, haloalkoxy, or cyano.
[0221] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00063##
and R.sup.5 is alkoxy or halo.
[0222] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00064##
and R.sup.5 is halo.
[0223] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00065##
and R.sup.5 is halo or cyano.
[0224] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00066##
and R.sup.5 is amido, halo, or cyano.
[0225] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00067##
and R.sup.5 is halo.
[0226] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00068##
and R.sup.5 is halo.
[0227] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00069##
and R.sup.5 is halo.
[0228] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00070##
is hydrogen, alkyl,
##STR00071##
m is 0, 1, or 2; and p is 0, 1, 2, or 3.
[0229] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00072##
is hydrogen.
[0230] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00073##
is alkyl.
[0231] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00074##
is methyl, ethyl, or propyl.
[0232] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00075##
[0233] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00076##
[0234] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00077##
[0235] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00078##
[0236] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00079##
[0237] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00080##
and R.sup.5 is halo, hydroxy, or alkoxy.
[0238] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00081##
and R.sup.5 is halo.
[0239] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00082##
[0240] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00083##
[0241] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00084##
[0242] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00085##
[0243] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00086##
[0244] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00087##
and R.sup.5 is alkoxycarbonyl.
[0245] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00088##
[0246] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00089##
and R.sup.2 is alkyl.
[0247] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00090##
and R.sup.2 is methyl.
[0248] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105##
##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110##
##STR00111## ##STR00112##
[0249] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula VII:
##STR00113##
[0250] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula VIII:
##STR00114##
[0251] Phthalazinone Series
[0252] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula IX:
##STR00115##
[0253] wherein, independently for each occurrence,
[0254] R.sup.2 is hydrogen or alkyl;
[0255] m is 0, 1, or 2;
##STR00116## [0256] is aryl, heteroaryl, amino, alkyl, cycloalkyl,
heterocycloalkyl, or aralkyl; wherein, any of the aforementioned
alkyl, aryl, heteroaryl, or aralkyl may be substituted with one or
more groups independently selected from the group consisting of
halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy,
alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,
acyloxy, silyl, alkylthio, sulfonate, sulfonyl, sulfonamido,
formyl, cyano, and isocyano.
[0257] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is hydrogen.
[0258] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is alkyl.
[0259] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein R.sup.2 is methyl, ethyl,
n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl.
[0260] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 0 or 1.
[0261] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 0.
[0262] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1.
[0263] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00117##
is alkyl, amino, benzyl,
##STR00118## ##STR00119##
p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R.sup.5 is halo,
azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,
haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl,
imino, amido, phosphonate, phosphinate, acyl, carboxyl,
alkoxycarbonyl, acyloxy, alkylthio, sulfonate, sulfonyl,
sulfonamido, formyl, cyano, or isocyano.
[0264] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00120##
is alkyl.
[0265] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00121##
is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or
t-butyl.
[0266] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00122##
is n-butyl.
[0267] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00123##
is s-butyl.
[0268] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00124##
is t-butyl.
[0269] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00125##
is amino.
[0270] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00126##
and R.sup.5 is amido, alkoxy, halo, haloalkyl, aryl, haloaryl,
alkyl, hydroxy, alkylthio, sulfonyl, haloalkoxy, or cyano.
[0271] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00127##
and R.sup.5 is alkoxy or halo.
[0272] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00128##
and R.sup.5 is halo.
[0273] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00129##
and R.sup.5 is halo or cyano.
[0274] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00130##
and R.sup.5 is amido, halo, or cyano.
[0275] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00131##
and R.sup.5 is halo.
[0276] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00132##
and R.sup.5 is amido.
[0277] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137##
##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142##
##STR00143## ##STR00144##
[0278] Naphthimidazole Series
[0279] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula X:
##STR00145##
[0280] wherein, independently for each occurrence,
[0281] m is 0, 1, 2, or 3;
[0282] X is absent, O, S, or NH; and
##STR00146## [0283] is aryl or heteroaryl; [0284] wherein, any of
the aforementioned aryl or heteroaryl, may be substituted with one
or more groups independently selected from the group consisting of
halo, azido, alkyl, haloalkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy,
alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, acyl, carboxyl, alkoxycarbonyl,
acyloxy, silyl, alkylthio, sulfonate, sulfonyl, sulfonamido,
formyl, cyano, and isocyano.
[0285] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein one occurrence of m is 0; and
one occurrence of m is 1.
[0286] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein one occurrence of m is 0; and
one occurrence of m is 2.
[0287] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein one occurrence of m is 0; and
one occurrence of m is 3.
[0288] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is O.
[0289] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is S.
[0290] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is NH.
[0291] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00147##
R.sup.5 is halo, azido, alkyl, haloalkyl, aralkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,
hydroxy, alkoxy, haloalkyloxy, aryloxy, heteroaryloxy, amino,
nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,
carboxyl, alkoxycarbonyl, acyloxy, alkylthio, sulfonate, sulfonyl,
sulfonamido, formyl, cyano, or isocyano; and q is 0 to 5
inclusive.
[0292] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00148##
and R.sup.5 is selected from the group consisting of halo, alkoxy,
haloalkyloxy, alkylthio, amido, and cyano.
[0293] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00149##
and R.sup.5 is halo.
[0294] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00150##
and R.sup.5 is chloro.
[0295] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00151##
and R.sup.5 is bromo.
[0296] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00152##
and R.sup.5 is alkoxy.
[0297] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00153##
and R.sup.5 is methoxy.
[0298] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00154##
and R.sup.5 is haloalkyloxy.
[0299] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00155##
and R.sup.5 is trifluoromethoxy.
[0300] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00156##
and R.sup.5 is alkylthio.
[0301] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00157##
and R.sup.5 is methylthio.
[0302] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00158##
and R.sup.5 is cyano.
[0303] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00159##
and R.sup.5 is halo.
[0304] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00160##
and R.sup.5 is chloro.
[0305] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00161##
and R.sup.5 is halo or cyano.
[0306] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00162##
and R.sup.5 is halo.
[0307] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00163##
and R.sup.5 is chloro.
[0308] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00164##
one instance of R.sup.5 is halo; and one instance of R.sup.5 is
cyano.
[0309] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00165##
one instance of R.sup.5 is halo; and one instance of R.sup.5 is
amido.
[0310] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00166##
one instance of R.sup.5 is chloro; and one instance of R.sup.5 is
cyano.
[0311] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00167##
and R.sup.5 is halo.
[0312] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00168##
and R.sup.5 is chloro.
[0313] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00169##
[0314] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00170##
[0315] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00171## ##STR00172##
[0316] Pyrazole Series
[0317] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula XI:
##STR00173##
[0318] wherein, independently for each occurrence,
[0319] m is 0, 1, or 2;
[0320] R.sup.2 is hydrogen or alkyl;
[0321] R.sup.3 is hydrogen or alkyl; and
##STR00174## [0322] is aryl or heteroaryl; [0323] wherein, any of
the aforementioned alkyl, aryl, or heteroaryl may be substituted
with one or more groups independently selected from the group
consisting of halo, azido, alkyl, haloalkyl, aralkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,
hydroxy, alkoxy, aryloxy, heteroaryloxy, amino, nitro, sulfhydryl,
imino, amido, phosphonate, phosphinate, acyl, carboxyl,
alkoxycarbonyl, acyloxy, silyl, alkylthio, sulfonate, sulfonyl,
sulfonamido, formyl, cyano, and isocyano.
[0324] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1.
[0325] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1 or 2; and R.sup.2 is
hydrogen.
[0326] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1 or 2; and R.sup.2 is
alkyl.
[0327] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1 or 2; and R.sup.2 is
methyl.
[0328] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00175##
is alkyl, amino, benzyl,
##STR00176## ##STR00177##
p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R.sup.5 is halo,
azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
heteroaralkyl, hydroxy, alkoxy, haloalkyloxy, aryloxy,
heteroaryloxy, amino, nitro, sulihydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, carboxylic acid,
acyloxy, alkylthio, sulfonate, sulfonyl, sulfonamido, formyl,
cyano, or isocyano.
[0329] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00178##
[0330] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00179##
[0331] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00180##
[0332] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00181##
[0333] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00182##
[0334] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00183##
[0335] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00184## ##STR00185## ##STR00186## ##STR00187##
##STR00188##
[0336] Urea Series
[0337] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula XII:
##STR00189##
[0338] wherein, independently for each occurrence,
[0339] m is 0, 1, or 2;
[0340] R.sup.2 is hydrogen or alkyl;
##STR00190## [0341] is aryl or heteroaryl; and
[0341] ##STR00191## [0342] is aryl or heteroaryl; [0343] wherein,
any of the aforementioned alkyl, aryl, or heteroaryl may be
substituted with one or more groups independently selected from the
group consisting of halo, azido, alkyl, haloalkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
heteroaralkyl, hydroxy, alkoxy, aryloxy, heteroaryloxy, amino,
nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, acyl,
carboxyl, alkoxycarbonyl, acyloxy, silyl, alkylthio, sulfonate,
sulfonyl, sulfonamido, formyl, cyano, and isocyano.
[0344] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 0.
[0345] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1.
[0346] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1 or 2; and R.sup.2 is
hydrogen.
[0347] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1 or 2; and R.sup.2 is
alkyl. In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00192##
is alkyl, amino, benzyl,
##STR00193## ##STR00194##
p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R.sup.5 is halo,
azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
heteroaralkyl, hydroxy, alkoxy, haloalkyloxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, carboxylic acid,
acyloxy, alkylthio, sulfonate, sulfonyl, sulfonamido, formyl,
cyano, or isocyano.
[0348] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00195##
[0349] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00196##
[0350] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00197##
[0351] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00198##
[0352] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00199##
[0353] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00200##
[0354] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00201##
[0355] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00202##
is alkyl, amino, benzyl,
##STR00203## ##STR00204##
p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R.sup.5 is halo,
azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,
heterocycloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,
haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulihydryl,
imino, amido, phosphonate, phosphinate, acyl, carboxyl,
alkoxycarbonyl, carboxylic acid, acyloxy, alkylthio, sulfonate,
sulfonyl, sulfonamido, formyl, cyano, oxime, or isocyano.
[0356] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00205##
[0357] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00206##
[0358] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00207##
[0359] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212##
##STR00213## ##STR00214## ##STR00215## ##STR00216## ##STR00217##
##STR00218## ##STR00219##
[0360] Benzoxazole Series
[0361] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, represented by
Formula XIII:
##STR00220##
[0362] wherein, independently for each occurrence,
[0363] X is absent or 0;
[0364] m is 0, 1, or 2;
[0365] R.sup.2 is hydrogen or alkyl;
##STR00221## [0366] is hydrogen, aryl, or heteroaryl; and
[0366] ##STR00222## [0367] is hydrogen, aryl, alkyl, or heteroaryl;
[0368] wherein, any of the aforementioned alkyl, aryl, or
heteroaryl may be substituted with one or more groups independently
selected from the group consisting of halo, azido, alkyl,
haloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy, aryloxy,
heteroaryloxy, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, acyl, carboxyl, alkoxycarbonyl, acyloxy, silyl,
alkylthio, sulfonate, sulfonyl, sulfonamido, formyl, cyano, and
isocyano.
[0369] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is absent.
[0370] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein X is O.
[0371] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 0.
[0372] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1.
[0373] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1; and R.sup.2 is
hydrogen.
[0374] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein m is 1; and R.sup.2 is
alkyl.
[0375] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00223##
is hydrogen, alkyl, amino, benzyl,
##STR00224## ##STR00225##
p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R.sup.5 is halo,
azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,
heterocycloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,
haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulihydryl,
imino, amido, phosphonate, phosphinate, acyl, carboxyl,
alkoxycarbonyl, carboxylic acid, acyloxy, alkylthio, sulfonate,
sulfonyl, sulfonamido, formyl, cyano, oxime, or isocyano.
[0376] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00226##
is hydrogen.
[0377] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00227##
[0378] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00228##
[0379] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00229##
[0380] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00230##
[0381] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00231##
[0382] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00232##
is hydrogen, alkyl, amino, benzyl,
##STR00233## ##STR00234##
p is 0, 1, 2, or 3; q is 0, 1, 2, 3, or 4; and R.sup.5 is halo,
azido, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,
heterocycloalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
heterocyclyl, aryl, heteroaryl, heteroaralkyl, hydroxy, alkoxy,
haloalkyloxy, aryloxy, heteroaryloxy, amino, nitro, sulihydryl,
imino, amido, phosphonate, phosphinate, acyl, carboxyl,
alkoxycarbonyl, carboxylic acid, acyloxy, alkylthio, sulfonate,
sulfonyl, sulfonamido, formyl, cyano, oxime, or isocyano.
[0383] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00235##
is hydrogen.
[0384] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00236##
is alkyl.
[0385] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00237##
[0386] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00238##
[0387] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt thereof, selected from the
group consisting of
##STR00239## ##STR00240##
[0388] General Considerations for Exemplary Compounds of the
Invention
[0389] When stereochemistry is not specifically indicated, the
compounds of the invention may contain one or more asymmetric
carbon atoms and thus may occur as racemates and racemic mixtures,
single enantiomers, diastereomeric mixtures and individual
diastereomers. All such isomeric forms of these compounds are
included in the present invention, unless expressly excluded. Each
stereogenic carbon may be of the R or S configuration.
[0390] In addition, the compounds of the invention described above
may be modified by appending appropriate functionalities to enhance
selective biological properties. Such modifications are known in
the art and include those which increase biological penetration
into a given biological compartment (e.g., blood, lymphatic system,
central nervous system), increase oral availability, increase
solubility to allow administration by injection, alter metabolism
and alter rate of excretion.
Pharmaceutical Compositions of the Invention
[0391] In certain embodiments, the invention relates to a
pharmaceutical composition, comprising a pharmaceutically
acceptable carrier, adjuvant, or vehicle; and any one of the
aforementioned compounds.
[0392] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an
antimicrobial agent.
[0393] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an antibiotic,
antifungal, or antiprotozoal agent.
[0394] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an antibiotic
agent selected from the group consisting of vancomycin,
metronidazole, amoxicillin, ciprofloxacin, doxycycline, gentamicin
and clindamycin.
[0395] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an antifungal
selected from the group consisting of terbinafine, flucytosine,
fluconazole, itraconazole, ketoconazole, voriconazole, nikkomycin
Z, caspofungin, micafungin (FK463), anidulafungin (LY303366),
amphotericin B (AmB), and nystatin.
[0396] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an
antiprotozoal agent selected from the group consisting of
eflornithine, furazolidone, melarsoprol, metronidazole, ornidazole,
paromomycin sulfate, pentamidine, pyrimethamine, and
tinidazole.
[0397] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an
immunosuppression agent.
[0398] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an
immunosuppression agent selected from the group consisting of
cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualin,
prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG,
interferon and mizoribine.
[0399] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an anti-cancer
agent.
[0400] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an anti-cancer
agent selected from the group consisting of cis-platin, actinomycin
D, doxorubicin, vincristine, vinblastine, etoposide, amsacrine,
mitoxantrone, teniposide, taxol, colchicine, cyclosporin A,
phenothiazines, interferon and thioxanthenes.
[0401] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an anti-viral
agent.
[0402] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an anti-viral
agent selected from the group consisting of cytovene, ganciclovir,
trisodium phosphonoformate, Ribavirin, d4T, ddI, AZT, and
acyclovir.
[0403] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an
anti-vascular hyperproliferative agent.
[0404] In certain embodiments, the invention relates to any one of
the aforementioned compositions, further comprising an
anti-vascular hyperproliferative selected from the group consisting
of HMG Co-A reductase inhibitors such as lovastatin, thromboxane A2
synthetase inhibitors, eicosapentanoic acid, ciprostene, trapidil,
ACE inhibitors, low molecular weight heparin, mycophenolic acid,
rapamycin and 5-(3'-pyridinylmethyl)benzofuran-2-carboxylate.
[0405] The compounds of the invention are defined to include
pharmaceutically acceptable salts or prodrugs thereof. A
"pharmaceutically acceptable salt or prodrug" means any
pharmaceutically acceptable salt, ester, salt of an ester, or other
derivative of a compound of the invention which, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. Particularly favored
prodrugs are those that increase the bioavailability of the
compounds of the invention when such compounds are administered to
a mammal (e.g., by allowing an orally administered compound to be
more readily absorbed into the blood) or which enhance delivery of
the parent compound to a biological compartment (e.g., the brain or
lymphatic system) relative to the parent species. Exemplary
prodrugs include derivatives where a group which enhances aqueous
solubility or active transport through the gut membrane is appended
to the structure of the compounds of the invention.
[0406] Pharmaceutically acceptable salts of the compounds of the
invention include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, succinate,
sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other
acids, such as oxalic, while not in themselves pharmaceutically
acceptable, may be employed in the preparation of salts useful as
intermediates in obtaining the compounds of the invention and their
pharmaceutically acceptable acid addition salts.
[0407] Salts derived from appropriate bases include alkali metal
(e.g., sodium), alkaline earth metal (e.g., magnesium), and
ammonium salts. This invention also envisions the quaternization of
any basic nitrogen-containing groups of the compounds disclosed
herein. Water or oil-soluble or dispersible products may be
obtained by such quaternization.
[0408] In certain embodiments, the invention relates to a
pharmaceutical composition, wherein the pharmaceutical composition
comprises any one of the aforementioned compounds or a
pharmaceutically acceptable salt thereof; an additional agent
selected from the group consisting of an immunosuppressant, an
anti-cancer agent, an anti-viral agent, antiinflammatory agent,
antifungal agent, antibiotic, and an anti-vascular
hyperproliferation compound; and any pharmaceutically acceptable
carrier, adjuvant or vehicle. In certain embodiments, the invention
relates to any one of the aforementioned pharmaceutical
compositions, wherein the pharmaceutical composition comprises any
one of the aforementioned compounds or a pharmaceutically
acceptable salt thereof; and a pharmaceutically acceptable carrier,
adjuvant or vehicle. In certain embodiments, the invention relates
to any one of the aforementioned pharmaceutical compositions,
wherein the pharmaceutical composition optionally comprises an
additional agent selected from the group consisting of an
immunosuppressant, an anti-cancer agent, an anti-viral agent,
antiinflammatory agent, antifungal agent, antibiotic, and an
anti-vascular hyperproliferation compound.
[0409] The term "pharmaceutically acceptable carrier or adjuvant"
refers to a carrier or adjuvant that may be administered to a
patient, together with a compound of this invention, and which does
not destroy the pharmacological activity thereof and is nontoxic
when administered in doses sufficient to deliver a therapeutic
amount of the compound.
[0410] Pharmaceutically acceptable carriers, adjuvants and vehicles
that may be used in the pharmaceutical compositions of the
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, self-emulsifying drug delivery systems
(SEDDS) such as d.alpha.-tocopherol polyethyleneglycol 1000
succinate, surfactants used in pharmaceutical dosage forms such as
Tweens or other similar polymeric delivery matrices, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat. Cyclodextrins such as .alpha.-, .beta.-, and
.gamma.-cyclodextrin, or chemically modified derivatives such as
hydroxyalkylcyclodextrins, including 2- and
3-hydroxypropyl-.beta.-cyclodextrins, or other solubilized
derivatives may also be advantageously used to enhance delivery of
any one of the aforementioned compounds.
[0411] The pharmaceutical compositions of the invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The pharmaceutical compositions of the invention may
contain any conventional non-toxic pharmaceutically-acceptable
carriers, adjuvants or vehicles. In some cases, the pH of the
formulation may be adjusted with pharmaceutically acceptable acids,
bases or buffers to enhance the stability of the formulated
compound or its delivery form. The term parenteral as used herein
includes subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intraarterial, intrasynovial, intrasternal,
intrathecal, intralesional and intracranial injection or infusion
techniques.
[0412] The pharmaceutical compositions may be in the form of a
sterile injectable preparation, for example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents (such as, for example, Tween 80) and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are mannitol, water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or diglycerides. Fatty acids,
such as oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant such as those described in Pharmacopeia Helvetica, Ph.
Helv., or a similar alcohol, or carboxymethyl cellulose or similar
dispersing agents which are commonly used in the formulation of
pharmaceutically acceptable dosage forms such as emulsions and or
suspensions. Other commonly used surfactants such as Tweens or
Spans and/or other similar emulsifying agents or bioavailability
enhancers which are commonly used in the manufacture of
pharmaceutically acceptable solid, liquid, or other dosage forms
may also be used for the purposes of formulation.
[0413] The pharmaceutical compositions of the invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, emulsions and aqueous
suspensions, dispersions and solutions. In the case of tablets for
oral use, carriers which are commonly used include lactose and corn
starch. Lubricating agents, such as magnesium stearate, are also
typically added. For oral administration in a capsule form, useful
diluents include lactose and dried corn starch. When aqueous
suspensions and/or emulsions are administered orally, the active
ingredient may be suspended or dissolved in an oily phase is
combined with emulsifying and/or suspending agents. If desired,
certain sweetening and/or flavoring and/or coloring agents may be
added.
[0414] The pharmaceutical compositions of the invention may also be
administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of the invention with a suitable non-irritating excipient
which is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax and polyethylene glycols.
[0415] Topical administration of the pharmaceutical compositions of
the invention is especially useful when the desired treatment
involves areas or organs readily accessible by topical application.
For application topically to the skin, the pharmaceutical
composition should be formulated with a suitable ointment
containing the active components suspended or dissolved in a
carrier. Carriers for topical administration of the compounds of
the invention include, but are not limited to, mineral oil, liquid
petroleum, white petroleum, propylene glycol, polyoxy-ethylene
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical composition can be formulated
with a suitable lotion or cream containing the active compound
suspended or dissolved in a carrier with suitable emulsifying
agents. Suitable carriers include, but are not limited to, mineral
oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,
cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The
pharmaceutical compositions of the invention may also be topically
applied to the lower intestinal tract by rectal suppository
formulation or in a suitable enema formulation.
Topically-transdermal patches are also included in this
invention.
[0416] The pharmaceutical compositions of the invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
[0417] Dosage levels of between about 0.01 and about 100 mg/kg body
weight per day, or between about 0.5 and about 75 mg/kg body weight
per day, of the IMPDH inhibitory compounds described herein are
useful in a monotherapy and/or in combination therapy for the
prevention and treatment of IMPDH-mediated disease or infection.
Typically, the pharmaceutical compositions of the invention will be
administered from about 1 to about 5 times per day or
alternatively, as a continuous infusion. Such administration can be
used as a chronic or acute therapy. The amount of active ingredient
that may be combined with the carrier materials to produce a single
dosage form will vary depending upon the host treated and the
particular mode of administration. A typical preparation will
contain from about 5% to about 95% active compound (w/w). Such
preparations contain from about 20% to about 80% active
compound.
[0418] When the compositions of the invention comprise a
combination of an IMPDH inhibitor of the invention and one or more
additional therapeutic or prophylactic agents, both the IMPDH
inhibitor and the additional agent should be present at dosage
levels of between about 10 to 100%, or between about 10 to 80% of
the dosage normally administered in a monotherapy regimen. The
additional agents may be administered separately, as part of a
multiple dose regimen, from the compounds of this invention.
Alternatively, those agents may be part of a single dosage form,
mixed together with the compounds of the invention in a single
composition.
[0419] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of the invention may
be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level, treatment should cease. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms.
[0420] As the skilled artisan will appreciate, lower or higher
doses than those recited above may be required. Specific dosage and
treatment regimens for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health status, sex, diet,
time of administration, rate of excretion, drug combination, the
severity and course of the infection, the patient's disposition to
the infection and the judgment of the treating physician.
[0421] In certain embodiments, the invention relates to a
pharmaceutical composition for treatment or prevention of a
protozoan infection, comprising a pharmaceutically acceptable
carrier, adjuvant or vehicle and at least one of the aforementioned
compounds, or a pharmaceutically acceptable salt or prodrug
thereof.
[0422] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, wherein said
protozoan infection is caused by a protozoan selected from the
group consisting of the genera Toxoplasma, Eimeria,
Cryptosporidium, Plasmodium, Babesia, Theileria, Neospora,
Sarcocystis, Giardia, Entamoeba, Trichomonas, Tritrichomonas,
Leishmania and Trypanosoma.
[0423] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, wherein said
protozoan infection is caused by a protozoan selected from the
genus Cryptosporidium.
[0424] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, wherein said
protozoan infection is caused by Cryptosporidium parvum.
[0425] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, wherein the
pharmaceutical composition further comprises an antimicrobial
agent, such as an antibiotic, antifungal, or antiprotozoal agent.
Examples of antibiotic agents include, but are not limited to,
vancomycin, metronidazole, amoxicillin, ciprofloxacin, doxycycline,
gentamicin and clindamycin. Examples of antifungal include, but are
not limited to, terbinafine, flucytosine, fluconazole,
itraconazole, ketoconazole, voriconazole, nikkomycin Z,
caspofungin, micafungin (FK463), anidulafungin (LY303366),
amphotericin B (AmB), and nystatin. Examples of antiprotozoal
agents include, but are not limited to, eflornithine, furazolidone,
melarsoprol, metronidazole, ornidazole, paromomycin sulfate,
pentamidine, pyrimethamine, and timidazole.
[0426] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, wherein the
pharmaceutical composition is used for treatment or prevention of
an IMPDH-mediated disease, and comprises a pharmaceutically
acceptable carrier, adjuvant or vehicle and at least one
aforementioned compound.
[0427] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, further comprising
an immunosuppression agent. Examples of additional
immunosuppression agents include, but are not limited to,
cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualin,
prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG,
interferon, and mizoribine.
[0428] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, further comprising
an anti-cancer agent. Examples of anti-cancer agents include, but
are not limited to, cis-platin, actinomycin D, doxorubicin,
vincristine, vinblastine, etoposide, amsacrine, mitoxantrone,
teniposide, taxol, colchicine, cyclosporin A, phenothiazines,
interferon, and thioxanthenes.
[0429] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, further comprising
an anti-viral agent. Examples of anti-viral agents include, but are
not limited to, cytovene, ganciclovir, trisodium phosphonoformate,
Ribavirin, d4T, ddI, AZT, and acyclovir.
[0430] In certain embodiments, the invention relates to any one of
the aforementioned pharmaceutical compositions, further comprising
an anti-vascular hyperproliferative agent. Examples of
anti-vascular hyperproliferative agents include, but are not
limited to, HMG Co-A reductase inhibitors such as lovastatin,
thromboxane A2 synthetase inhibitors, eicosapentanoic acid,
ciprostene, trapidil, ACE inhibitors, low molecular weight heparin,
mycophenolic acid, rapamycin, and
5-(3'-pyridinylmethyl)benzofuran-2-carboxylate.
Selected Methods of the Invention
[0431] In certain embodiments, the invention relates to a method of
killing or inhibiting the growth of a microbe, comprising the step
of contacting said microbe with an effective amount of any one of
the aforementioned compounds.
[0432] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbe is a protozoon, a
bacterium, or a fungus.
[0433] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbe is a protozoon or
a bacterium selected from the group consisting of the genera
Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,
Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,
Tritrichomonas, Leishmania, Trypanosoma, Helicobacter, Borrelia,
Salmonella, Shigella, Yersinia, Streptococcus, Campylobacter,
Arcobacter, Bacteroides, Fusobacterium, Burkholderia, Clostridia,
Neisseria, Mycobacterium, and Acinetobacter.
[0434] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbe is a protozoon;
and said protozoon is selected from the group consisting of the
genera Toxoplasma, Eimeria, Cryptosporidium, Plasmodium, Babesia,
Theileria, Neospora, Sarcocystis, Giardia, Entamoeba, Trichomonas,
Tritrichomonas, Leishmania and Trypanosoma.
[0435] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said protozoon is selected from
the genus Cryptosporidium.
[0436] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said protozoon is
Cryptosporidium parvum.
[0437] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbe is a bacterium;
and said bacterium is selected from the group consisting of the
genera Helicobacter, Borrelia, Salmonella, Shigella, Yersinia,
Streptococcus, Campylobacter, Arcobacter, Bacteroides,
Fusobacterium, Burkholderia, Clostridia, Neisseria, Mycobacterium,
and Acinetobacter.
[0438] In certain embodiments, the invention relates to a method of
treating or preventing a microbial infection in a mammal,
comprising the step of administering to a mammal in need thereof a
therapeutically effective amount of any one of the aforementioned
compounds.
[0439] In certain embodiments, the invention relates to a method of
treating or preventing a parasitic infection in a mammal comprising
the step of administering to a mammal in need thereof a
therapeutically effective amount of any one of the aforementioned
compounds.
[0440] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbial infection is
caused by a protozoon, a bacterium, or a fungus.
[0441] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbial infection is
caused by a protozoon or a bacterium selected from the group
consisting of the genera Toxoplasma, Eimeria, Cryptosporidium,
Plasmodium, Babesia, Theileria, Neospora, Sarcocystis, Giardia,
Entamoeba, Trichomonas, Tritrichomonas, Leishmania, Trypanosoma,
Helicobacter, Borrelia, Salmonella, Shigella, Yersinia,
Streptococcus, Campylobacter, Arcobacter, Bacteroides,
Fusobacterium, Burkholderia, Clostridia, Neisseria, Mycobacterium,
and Acinetobacter.
[0442] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbial infection is
caused by a protozoon; and said protozoon is selected from the
group consisting of the genera Toxoplasma, Eimeria,
Cryptosporidium, Plasmodium, Babesia, Theileria, Neospora,
Sarcocystis, Giardia, Entamoeba, Trichomonas, Tritrichomonas,
Leishmania and Trypanosoma.
[0443] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said protozoon is selected from
the genus Cryptosporidium.
[0444] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbial infection is
caused by Cryptosporidium parvum.
[0445] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said microbe is a bacterium;
and said bacterium is selected from the group consisting of the
genera Helicobacter, Borrelia, Salmonella, Shigella, Yersinia,
Streptococcus, Campylobacter, Arcobacter, Bacteroides,
Fusobacterium, Burkholderia, Clostridia, Neisseria, Mycobacterium,
and Acinetobacter.
[0446] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the step of
co-administering to a mammal in need thereof a therapeutically
effective amount of an antimicrobial agent.
[0447] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said antimicrobial agent is an
antibiotic. In certain embodiments, the invention relates to any
one of the aforementioned methods, wherein said antimicrobial agent
is an antibiotic. In certain embodiments, the invention relates to
any one of the aforementioned methods, wherein said antibiotic
agent is selected from the group consisting of vancomycin,
metronidazole, amoxicillin, ciprofloxacin, doxycycline, gentamicin,
and clindamycin.
[0448] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said antimicrobial agent is an
antifungal. In certain embodiments, the invention relates to any
one of the aforementioned methods, wherein said antifungal agent is
selected from the group consisting of terbinafine, flucytosine,
fluconazole, itraconazole, ketoconazole, voriconazole, nikkomycin
Z, caspofungin, micafungin (FK463), anidulafungin (LY303366),
amphotericin B (AmB), and nystatin.
[0449] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein said antimicrobial agent is an
antiparasitic. In certain embodiments, the invention relates to any
one of the aforementioned methods, wherein said antiparasitic agent
is selected from the group consisting of eflornithine,
furazolidone, melarsoprol, metronidazole, ornidazole, paromomycin
sulfate, pentamidine, pyrimethamine, and timidazole.
[0450] In certain embodiments, the invention relates to a method of
treating or preventing an IMPDH-mediated disease in a mammal,
comprising the step of administering to a mammal in need thereof a
therapeutically effective amount of any one of the aforementioned
compounds. If the pharmaceutical composition only comprises the
IMPDH inhibitor of the invention as the active component, such
methods may additionally comprise the step of administering to a
mammal in need thereof a therapeutically effective amount of an
agent selected from an antiinflammatory agent, immunosuppressant,
an anti-cancer agent, an anti-viral agent, or an anti-vascular
hyperproliferation compound. Such additional agents may be
administered to the mammal prior to, concurrently with, or
following the administration of the IMPDH inhibitor
composition.
[0451] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the IMPDH-mediated disease is
transplant rejection, graft versus host disease, rheumatoid
arthritis, multiple sclerosis, juvenile diabetes, asthma,
inflammatory bowel disease, Crohn's disease, ulcerative colitus,
lupus, diabetes, mellitus myasthenia gravis, psoriasis, dermatitis,
eczema, seborrhea, pulmonary inflammation, eye uveitis, hepatitis,
Grave's disease, Hashimoto's thyroiditis, Behcet's or Sjorgen's
syndrome, pernicious or immunohaemolytic anemia, idiopathic adrenal
insufficiency, polyglandular autoimmune syndrome,
glomerulonephritis, scleroderma, lichen planus, viteligo,
autoimmune thyroiditis, alveolitis, HTLV-1, HTLV-2, HIV-1, HIV-2,
nasopharyngeal carcinoma virus, HBV, HCV, HGV, yellow fever virus,
dengue fever virus, Japanese encephalitis virus, human papilloma
virus, Epstein-Barr, cytomegaloviruses, Herpes Simplex Type 1,
Herpes Simplex Type 2, Herpes Simplex Type 6, restenosis, stenosis,
artherosclerosis, lymphoma, leukemia, osteoarthritis, acute
pancreatitis, chronic pancreatitis, asthma, or adult respiratory
distress syndrome.
[0452] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the step of
co-administering to a mammal in need thereof a therapeutically
effective amount of an agent selected from the group consisting of
an antiinflammatory agent, immunosuppressant, an anti-cancer agent,
an anti-viral agent, and an anti-vascular hyperproliferation
compound.
[0453] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the method is useful in
suppressing an immune response in a mammal. Such methods are useful
in treating or preventing diseases, including, transplant rejection
(e.g., kidney, liver, heart, lung, pancreas (islet cells), bone
marrow, cornea, small bowel and skin allografts and heart valve
xenografts), graft versus host disease, and autoimmune diseases,
such as rheumatoid arthritis, multiple sclerosis, juvenile
diabetes, asthma, inflammatory bowel disease (Crohn's disease,
ulcerative colitus), lupus, diabetes, mellitus myasthenia gravis,
psoriasis, dermatitis, eczema, seborrhea, pulmonary inflammation,
eye uveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis,
Behcet's or Sjorgen's syndrome (dry eyes/mouth), pernicious or
immunohaemolytic anemia, idiopathic adrenal insufficiency,
polyglandular autoimmune syndrome, glomerulonephritis, scleroderma,
lichen planus, viteligo (depigmentation of the skin), autoimmune
thyroiditis, and alveolitis.
[0454] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the method comprises the step
of administering to the mammal a composition comprising any one of
the aforementioned compounds and a pharmaceutically acceptable
adjuvant. In certain embodiments, the invention relates to any one
of the aforementioned methods, further comprising the step of
administering to a mammal in need thereof a composition comprising
an additional immunosuppressant and a pharmaceutically acceptable
adjuvant.
[0455] In certain embodiments, the invention relates to any one of
the aforementioned methods, comprising the step of administering to
a mammal in need thereof a composition comprising a compound of the
invention; an additional immunosuppressive agent and a
pharmaceutically acceptable adjuvant.
[0456] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the method is useful for
inhibiting viral replication in a mammal. Such methods are useful
in treating or preventing, DNA and RNA viral diseases caused by,
for example, HTLV-1 and HTLV-2, HIV-1 and HIV-2, nasopharyngeal
carcinoma virus, HBV, HCV, HGV, yellow fever virus, dengue fever
virus, Japanese encephalitis virus, human papilloma virus,
rhinoviruses and Herpes viruses, such as Epstein-Barr,
cytomegaloviruses and Herpes Simplex, Types 1 and 2, or Type 6. See
U.S. Pat. No. 5,380,879 (incorporated by reference).
[0457] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the method comprises the step
of administering to the mammal a composition comprising any one of
the aforementioned compounds, and a pharmaceutically acceptable
adjuvant. In certain embodiments, the invention relates to any one
of the aforementioned methods, further comprising the step of
administering to a mammal in need thereof a composition comprising
an additional anti-viral agent and a pharmaceutically acceptable
adjuvant.
[0458] In certain embodiments, the invention relates to any one of
the aforementioned methods, comprising the step of administering to
a mammal in need thereof a composition comprising any one of the
aforementioned compounds; an additional anti-viral agent and a
pharmaceutically acceptable adjuvant.
[0459] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the method is useful for
inhibiting vascular cellular hyperproliferation in a mammal. Such
methods are useful in treating or preventing diseases, including,
restenosis, stenosis, artherosclerosis and other hyperproliferative
vascular disease.
[0460] In certain embodiments, the invention relates to any one of
the aforementioned methods, comprising the step of administering to
the mammal a composition comprising any one of the aforementioned
compounds, and a pharmaceutically acceptable adjuvant. In certain
embodiments, the invention relates to any one of the aforementioned
methods, further comprising the step of administering to a mammal
in need thereof a therapeutically effective amount of a composition
comprising an additional anti-vascular hyperproliferative agent and
a pharmaceutically acceptable adjuvant.
[0461] In certain embodiments, the invention relates to any one of
the aforementioned methods, comprising the step of administering to
a mammal in need thereof a therapeutically effective amount of a
composition comprising any one of the aforementioned compounds; an
additional anti-vascular hyperproliferative agent and a
pharmaceutically acceptable adjuvant.
[0462] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the method is useful for
inhibiting tumors and cancer in a mammal. Such methods are useful
in treating or preventing diseases, including, tumors and
malignancies, such as lymphoma, leukemia and other forms of
cancer.
[0463] In certain embodiments, the invention relates to any one of
the aforementioned methods, comprising the step of administering to
the mammal a therapeutically effective amount of a composition
comprising any one of the aforementioned compounds, and a
pharmaceutically acceptable adjuvant. In certain embodiments, the
invention relates to any one of the aforementioned methods, further
comprising the step of administering to a mammal in need thereof a
therapeutically effective amount of a composition comprising an
additional anti-tumor or anti-cancer agent and a pharmaceutically
acceptable adjuvant.
[0464] In certain embodiments, the invention relates to any one of
the aforementioned methods, comprising the step of administering to
a mammal in need thereof a composition comprising any one of the
aforementioned compounds; a therapeutically effective amount of an
additional anti-tumor or anti-cancer agent and a pharmaceutically
acceptable adjuvant.
[0465] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the method is useful for
inhibiting inflammation and inflammatory diseases in a mammal. Such
methods are useful in treating or preventing diseases, including,
osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma
and adult respiratory distress syndrome.
[0466] In certain embodiments, the invention relates to any one of
the aforementioned methods, comprising the step of administering to
the mammal a composition comprising a therapeutically effective
amount of any one of the aforementioned compounds, and a
pharmaceutically acceptable adjuvant. In certain embodiments, the
invention relates to any one of the aforementioned methods, further
comprising the step of administering to a mammal in need thereof a
composition comprising a therapeutically effective amount of an
antiinflammatory agent and a pharmaceutically acceptable
adjuvant.
[0467] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the mammal is a primate, a
bovine, an ovine, an equine, a porcine, a rodent, a feline, a
mustelid, or a canine
[0468] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the mammal is a primate.
[0469] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the mammal is a human.
DEFINITIONS
[0470] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0471] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0472] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0473] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e., "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0474] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0475] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0476] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0477] The term "heteroatom" is art-recognized and refers to an
atom of any element other than carbon or hydrogen. Illustrative
heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and
selenium.
[0478] The term "alkyl" is art-recognized, and includes saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In certain embodiments, a straight chain or branched chain
alkyl has about 80 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.80 for straight chain, C.sub.3-C.sub.80 for branched
chain), and alternatively, about 30 or fewer. Likewise, cycloalkyls
have from about 3 to about 10 carbon atoms in their ring structure,
and alternatively about 5, 6 or 7 carbons in the ring structure. As
used herein, "fluoroalkyl" denotes an alkyl where one or more
hydrogens have been replaced with fluorines.
[0479] Unless the number of carbons is otherwise specified, "lower
alkyl" refers to an alkyl group, as defined above, but having from
one to about ten carbons, alternatively from one to about six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths.
[0480] The term "aralkyl" is art-recognized and refers to an alkyl
group substituted with an aryl group (e.g., an aromatic or
heteroaromatic group).
[0481] The terms "alkenyl" and "alkynyl" are art-recognized and
refer to unsaturated aliphatic groups analogous in length and
possible substitution to the alkyls described above, but that
contain at least one double or triple bond respectively.
[0482] The term "aryl" is art-recognized and refers to 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, naphthalene, anthracene,
pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,
triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine,
and the like. Those aryl groups having heteroatoms in the ring
structure may also be referred to as "aryl heterocycles" or
"heteroaromatics." The aromatic ring may be substituted at one or
more ring positions with such substituents as described herein, for
example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulihydryl, imino,
amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl,
alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,
heterocyclyl, aromatic or heteroaromatic moieties, trifluoromethyl,
cyano, or the like. The term "aryl" also includes polycyclic ring
systems having two or more cyclic rings in which two or more
carbons are common to two adjoining rings (the rings are "fused
rings") wherein at least one of the rings is aromatic, e.g., the
other cyclic rings may be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocyclyls.
[0483] The terms ortho, meta and para are art-recognized and refer
to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For
example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene
are synonymous.
[0484] The terms "heterocyclyl", "heteroaryl", or "heterocyclic
group" are art-recognized and refer to 3- to about 10-membered ring
structures, alternatively 3- to about 7-membered rings, whose ring
structures include one to four heteroatoms. Heterocycles may also
be polycycles. Heterocyclyl groups include, for example, thiophene,
thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,
phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole,
isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,
isoindole, indole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,
cinnoline, pteridine, carbazole, carboline, phenanthridine,
acridine, pyrimidine, phenanthroline, phenazine, phenarsazine,
phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,
thiolane, oxazole, piperidine, piperazine, morpholine, lactones,
lactams such as azetidinones and pyrrolidinones, sultams, sultones,
and the like. The heterocyclic ring may be substituted at one or
more positions with such substituents as described above, as for
example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, trifluoromethyl, cyano, or the like.
[0485] The terms "polycyclyl" or "polycyclic group" are
art-recognized and refer to two or more rings (e.g., cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which
two or more carbons are common to two adjoining rings, e.g., the
rings are "fused rings". Rings that are joined through non-adjacent
atoms are termed "bridged" rings. Each of the rings of the
polycycle may be substituted with such substituents as described
above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,
phosphonate, phosphinate, carbonyl, carboxyl, silyl, alkylthio,
sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, trifluoromethyl, cyano, or the like.
[0486] The term "carbocycle" is art-recognized and refers to an
aromatic or non-aromatic ring in which each atom of the ring is
carbon.
[0487] The terms "monocyclic," "bicyclic," or "tricyclic" ring
systems refers to 5 or 6 member monocyclic rings, 8, 9 and 10
membered bicyclic ring structures, and 11, 12, 13 and 14 membered
tricyclic ring structures, wherein each bond in each ring may be
possess any degree of saturation that is chemically feasible. When
such structures contain substituents, those substituents may be at
any position of the ring system, unless otherwise specified. As
specified, such ring systems may optionally comprise up to 4
heteroatoms selected from N, O or S. Those heteroatoms may replace
any carbon atoms in these ring systems as long as the resulting
compound is chemically stable.
[0488] The term "monocyclic" ring system, as used herein, includes
saturated, partially unsaturated and fully unsaturated ring
structures. The term "bicyclic" ring system, as used herein,
includes systems wherein each ring is independently saturated,
partially unsaturated and fully unsaturated. Examples of monocyclic
and bicyclic ring systems useful in the compounds of the invention
include, but are not limited to, cyclopentane, cyclopentene,
indane, indene, cyclohexane, cyclohexene, cyclohexadiene, benzene,
tetrahydronaphthalene, decahydronaphthalene, naphthalene, pyridine,
piperidine, pyridazine, pyrimidine, pyrazine, 1,2,3-triazine,
1,2,4-triazine, 1,3,5-triazine, 1,2,3,4-tetrazine,
1,2,4,5-tetrazine, 1,2,3,4-tetrahydroquinoline, quinoline,
1,2,3,4-tetrahydroisoquinoline, isoquinoline, cinnoline,
phthalazine, quinazoline, quinoxaline, 1,5-naphthyridine,
1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine,
2,6-naphthyridine, 2,7-naphthyridine, pteridine, acridine,
phenazine, 1,10-phenantroline, dibenzopyrans, 1-benzopyrans,
phenothiazine, phenoxazine, thianthrene, dibenzo-p-dioxin,
phenoxathiin, phenoxthionine, morpholine, thiomorpholine,
tetrahydropyran, pyran, benzopyran, 1,4-dioxane, 1,3-dioxane,
dihydropyridine, dihydropyran, 1-pyrindine, quinuclidine,
triazolopyridine, .beta.-carboline, indolizine, quinolizidine,
tetrahydronaphthyridine, diazaphenanthrene, thiopyran,
tetrahydrothiopyran, benzodioxane, furan, benzofuran,
tetrahydrofuran, pyrrole, indole, thiophene, benzothiophene,
carbazole, pyrrolidine, pyrazole, isoxazole, isothiazole,
imidazole, oxazole, thiazole, 1,2,3-triazole, 1,2,4-triazole,
1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4 oxadiazole,
1,2,5-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole,
1,3,4-thiadiazole, 1,2,5 thiadiazole, tetrazole, benzothiazole,
benzoxazole, benzotriazole, benzimidazole, benzopyrazole,
benzisothiazole, benzisoxazole and purine.
[0489] Additional monocyclic and bicyclic structures falling within
the above description may be found in A. R. Katritzky, and C. W.
Rees, eds. "Comprehensive Heterocyclic Chemistry: Structure,
Reactions, Synthesis and Use of Heterocyclic Compounds, Vol. 1-8,"
Pergamon Press, NY (1984), the disclosure of which is herein
incorporated by reference.
[0490] It should be understood that heterocycles may be attached to
the rest of the compound by any atom of the heterocycle which
results in the creation of a stable structure.
[0491] The term "ring atom", as used herein, refers to a backbone
atom that makes up the ring. Such ring atoms are selected from C,
N, O or S and are bound to 2 or 3 other such ring atoms (3 in the
case of certain ring atoms in a bicyclic ring system). The term
"ring atom" does not include hydrogen.
[0492] The term "nitro" is art-recognized and refers to --NO.sub.2;
the term "halogen" is art-recognized and refers to --F, --Cl, --Br
or --I; the term "sulfhydryl" is art-recognized and refers to --SH;
the term "hydroxyl" means --OH; and the term "sulfonyl" is
art-recognized and refers to --SO.sub.2.sup.-. "Halide" designates
the corresponding anion of the halogens, and "pseudohalide" has the
definition set forth on page 560 of "Advanced Inorganic Chemistry"
by Cotton and Wilkinson, that is, for example, monovalent anionic
groups sufficiently electronegative to exhibit a positive Hammett
sigma value at least equaling that of a halide (e.g., CN, OCN, SCN,
SeCN, TeCN, N.sub.3, and C(CN).sub.3).
[0493] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
may be represented by the general formulas:
##STR00241##
wherein R50, R51, R52 and R53 each independently represent a
hydrogen, an alkyl, an alkenyl, --(CH.sub.2).sub.m--R61, or R50 and
R51 or R52, taken together with the N atom to which they are
attached complete a heterocycle having from 4 to 8 atoms in the
ring structure; R61 represents an aryl, a cycloalkyl, a
cycloalkenyl, a heterocycle or a polycycle; and m is zero or an
integer in the range of 1 to 8. In other embodiments, R50 and R51
(and optionally R52) each independently represent a hydrogen, an
alkyl, an alkenyl, or --(CH.sub.2).sub.m--R61. Thus, the term
"alkylamine" includes an amine group, as defined above, having a
substituted or unsubstituted alkyl attached thereto, i.e., at least
one of R50 and R51 is an alkyl group.
[0494] The term "acylamino" is art-recognized and refers to a
moiety that may be represented by the general formula:
##STR00242##
wherein R50 is as defined above, and R54 represents a hydrogen, an
alkyl, an alkenyl or --(CH.sub.2).sub.m--R61, where m and R61 are
as defined above.
[0495] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that may be represented by the
general formula:
##STR00243##
wherein R50 and R51 are as defined above. Certain embodiments of
the amide in the present invention will not include imides which
may be unstable.
[0496] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In certain
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R61, wherein m and R61 are defined above.
Representative alkylthio groups include methylthio, ethyl thio, and
the like.
[0497] The term "carboxyl" is art recognized and includes such
moieties as may be represented by the general formulas:
##STR00244##
wherein X50 is a bond or represents an oxygen or a sulfur, and R55
and R56 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R61 or a pharmaceutically acceptable salt, R56
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R61, where m and R61 are defined above. Where
X50 is an oxygen and R55 or R56 is not hydrogen, the formula
represents an "ester." Where X50 is an oxygen, and R55 is as
defined above, the moiety is referred to herein as a carboxyl
group, and particularly when R55 is a hydrogen, the formula
represents a "carboxylic acid." Where X50 is an oxygen, and R56 is
hydrogen, the formula represents a "formate." In general, where the
oxygen atom of the above formula is replaced by sulfur, the formula
represents a "thiocarbonyl" group. Where X50 is a sulfur and R55 or
R56 is not hydrogen, the formula represents a "thioester." Where
X50 is a sulfur and R55 is hydrogen, the formula represents a
"thiolcarboxylic acid." Where X50 is a sulfur and R56 is hydrogen,
the formula represents a "thioformate." On the other hand, where
X50 is a bond, and R55 is not hydrogen, the above formula
represents a "ketone" group. Where X50 is a bond, and R55 is
hydrogen, the above formula represents an "aldehyde" group.
[0498] The term "carbamoyl" refers to --O(C.dbd.O)NRR', where R and
R' are independently H, aliphatic groups, aryl groups or heteroaryl
groups.
[0499] The term "oxo" refers to a carbonyl oxygen (.dbd.O).
[0500] The terms "oxime" and "oxime ether" are art-recognized and
refer to moieties that may be represented by the general
formula:
##STR00245##
wherein R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,
aralkyl, or --(CH.sub.2).sub.m--R61. The moiety is an "oxime" when
R is H; and it is an "oxime ether" when R is alkyl, cycloalkyl,
alkenyl, alkynyl, aryl, aralkyl, or --(CH.sub.2).sub.m--R61.
[0501] The terms "alkoxyl" or "alkoxy" are art-recognized and refer
to an alkyl group, as defined above, having an oxygen radical
attached thereto. Representative alkoxyl groups include methoxy,
ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two
hydrocarbons covalently linked by an oxygen. Accordingly, the
substituent of an alkyl that renders that alkyl an ether is or
resembles an alkoxyl, such as may be represented by one of
--O-alkyl, --O-alkenyl, --O-alkynyl, --O--(CH.sub.2).sub.m--R61,
where m and R61 are described above.
[0502] The term "sulfonate" is art recognized and refers to a
moiety that may be represented by the general formula:
##STR00246##
in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or
aryl.
[0503] The term "sulfate" is art recognized and includes a moiety
that may be represented by the general formula:
##STR00247##
in which R57 is as defined above.
[0504] The term "sulfonamido" is art recognized and includes a
moiety that may be represented by the general formula:
##STR00248##
in which R50 and R56 are as defined above.
[0505] The term "sulfamoyl" is art-recognized and refers to a
moiety that may be represented by the general formula:
##STR00249##
in which R50 and R51 are as defined above.
[0506] The term "sulfonyl" is art-recognized and refers to a moiety
that may be represented by the general formula:
##STR00250##
in which R58 is one of the following: hydrogen, alkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.
[0507] The term "sulfoxide" is art-recognized and refers to a
moiety that may be represented by the general formula:
##STR00251##
in which R58 is defined above.
[0508] The term "phosphoryl" is art-recognized and may in general
be represented by the formula:
##STR00252##
wherein Q50 represents S or O, and R59 represents hydrogen, a lower
alkyl or an aryl. When used to substitute, e.g., an alkyl, the
phosphoryl group of the phosphorylalkyl may be represented by the
general formulas:
##STR00253##
wherein Q50 and R59, each independently, are defined above, and Q51
represents O, S or N. When Q50 is S, the phosphoryl moiety is a
"phosphorothioate."
[0509] The term "phosphoramidite" is art-recognized and may be
represented in the general formulas:
##STR00254##
wherein Q51, R50, R51 and R59 are as defined above.
[0510] The term "phosphonamidate" is art-recognized and may be
represented in the general formulas:
##STR00255##
wherein Q51, R50, R51 and R59 are as defined above, and R60
represents a lower alkyl or an aryl.
[0511] Analogous substitutions may be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0512] The term "selenoalkyl" is art-recognized and refers to an
alkyl group having a substituted seleno group attached thereto.
Exemplary "selenoethers" which may be substituted on the alkyl are
selected from one of --Se-alkyl, --Se-alkenyl, --Se-alkynyl, and
--Se--(CH.sub.2).sub.m--R61, m and R61 being defined above.
[0513] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0514] The definition of each expression, e.g., alkyl, m, n, and
the like, when it occurs more than once in any structure, is
intended to be independent of its definition elsewhere in the same
structure.
[0515] The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations.
[0516] Certain compounds contained in compositions of the present
invention may exist in particular geometric or stereoisomeric
forms. In addition, polymers of the present invention may also be
optically active. The present invention contemplates all such
compounds, including cis- and trans-isomers, R- and S-enantiomers,
diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures
thereof, and other mixtures thereof, as falling within the scope of
the invention. Additional asymmetric carbon atoms may be present in
a substituent such as an alkyl group. All such isomers, as well as
mixtures thereof, are intended to be included in this
invention.
[0517] If, for instance, a particular enantiomer of compound of the
present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts are formed with an appropriate
optically-active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0518] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, or other
reaction.
[0519] The term "substituted" is also contemplated to include all
permissible substituents of organic compounds. In a broad aspect,
the permissible substituents include acyclic and cyclic, branched
and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic substituents of organic compounds. Illustrative
substituents include, for example, those described herein above.
The permissible substituents may be one or more and the same or
different for appropriate organic compounds. For purposes of this
invention, the heteroatoms such as nitrogen may have hydrogen
substituents and/or any permissible substituents of organic
compounds described herein which satisfy the valences of the
heteroatoms. This invention is not intended to be limited in any
manner by the permissible substituents of organic compounds.
[0520] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, "Handbook of Chemistry and Physics", 67th Ed.,
1986-87, inside cover.
[0521] The term "treating" as used herein refers to the alleviation
of symptoms of a particular disorder in a patient or the
improvement of an ascertainable measurement associated with a
particular disorder. As used herein, the term "patient" refers to a
mammal, including a human.
[0522] While several embodiments of the present invention are
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
EXEMPLIFICATION
[0523] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
Example 1
General Synthetic Strategy for Triazole Inhibitors
[0524] Various 1,2,3-triazole analogs were prepared according to
FIG. 7. The common intermediate in the synthesis of these
derivatives was propargyl ether 43. This intermediate was prepared
using three different routes. In the first route (R'=Me or H), 40
was alkylated utilizing either a Mitsunobu reaction with a
propargyl alcohol or by treatment with propargyl bromide in the
presence of potassium carbonate. The route using the Mitsunobu
reaction also afforded enantiomerically enriched ethers stating
with (S)- or (R)-but-3-yn-2-ol. When R.sup.1=i-Pr and
R.sup.2=CO.sub.2H, the acid 41 (prepared via FIG. 8) was reduced to
the corresponding alcohol with LiAlH.sub.4 and then oxidized to
aldehyde 42. When R.sup.1=Et, R.sup.2=CO.sub.2Et, the ester 41
(prepared via FIG. 8) was reduced directly to aldehyde 42 with
DIBAL in THF at -78.degree. C. Aldehyde 42 was converted to the
corresponding alkyne. Ether 43 was converted to 1,2,3-triazole 44
in the presence of an aryl azide and Cut In the case of N-oxide
derivatives, 43 (X or Y.dbd.N) was first treated with m-CPBA. The
N-oxide of 43 was then converted to 44 (X or
Y.dbd.N.sup.+--O.sup.-).
Example 2
Synthesis of ethyl .alpha.-bromocyclopropaneacetate (46,
R=c-Pr)
[0525] A flame dried two-neck round bottom flask fitted with a
reflux condenser and N.sub.2 outlet was charged with anhydrous THF,
freshly activated Mg (120 mg, 4.95 mmol) and a catalytic amount of
iodine. A small portion of cyclopropyl bromide dissolved in THF was
added. After initiation of reflux, the reaction mixture was cooled
to -20.degree. C. and the remaining cyclopropyl bromide (500 mg,
4.13 mmol) was gradually added. After 30 min a freshly distilled
solution of glyoxylate 45 (549 mg, 5.37 mmol) in THF was added over
a 10 min period and the resulting solution was stirred at
-20.degree. C. for 2 h before being quenched with a small amount of
water. After 10 min the reaction mixture was further diluted with
water (50 mL) and extracted with ethyl acetate (3.times.50 mL). The
organic extracts were combined, dried over anhydrous MgSO.sub.4,
filtered, concentrated in vacuo and purified by column
chromatography eluting with ethyl acetate/n-hexane (a gradient of
10-20%) to furnish ethyl .alpha.-hydroxycyclopropaneacetate (422
mg, 71%) as a viscous oil. The oil (350 mg, 2.43 mmol) was
dissolved in anhydrous DCM and cooled to 0.degree. C. Then
Ph.sub.3P (2.04 gm, 7.78 mmol) was added followed by CBr.sub.4
(1.20 gm, 3.64 mmol). The reaction mixture was stirred at 0.degree.
C. for 2 h and then concentrated in vacuo. The Ph.sub.3PO was
precipitated by addition of n-hexane and removed by filtration. The
crude reaction mixture was purified by flash column chromatography
to furnish ethyl .alpha.-bromocyclopropaneacetate (46, R=c-Pr):
(311 mg, 62% yield).
Example 3
General Procedure for the Synthesis of 2-(1-naphthalenyloxy)acetic
acids (41)
[0526] Exemplified for 2-cyclopropyl-2-(1-naphthalenyloxy)acetic
acid (41, R=c-Pr): To a solution of 1-naphthol (170 mg, 1.18 mmol)
in anhydrous DMF (10 mL) was added K.sub.2CO.sub.3 (510 mg, 3.53
mmol) and ethyl .alpha.-bromocyclopropaneacetate (295 mg, 1.41
mmol). The mixture was stirred at room temperature for 2 h and then
diluted with water (50 mL) and then extracted with ethyl acetate
(3.times.50 mL). The organic extracts were combined, washed with
brine, dried over anhydrous MgSO.sub.4, filtered, concentrated in
vacuo and purified by flash column chromatography using a mixture
of ethyl acetate and n-hexane (1:9) to furnish ethyl
2-cyclopropyl-2-(1-naphthalenyloxy)acetate (8, R=c-Pr, 296 mg, 93%)
as a white solid. The ester (250 mg, 0.92 mmol) was dissolved in 20
mL THF: H.sub.2O (2:1) and then 3M NaOH (111 mg, 2.77 mmol) was
added. The reaction was heated at 80.degree. C. for 6 h. After
cooling, the reaction mixture was quenched with 1N HCL to a pH
.about.7 and then extracted with chloroform. The organic extract
was dried over anhydrous MgSO.sub.4, filtered, concentrated in
vacuo and purified by flash column chromatography eluting with a
mixture of ethyl acetate/n-hexane (a gradient of 2:1) to furnish
2-cyclopropyl-2-(1-naphthalenyloxy)acetic acid (41, R=c-Pr) (136
mg, 61%) as a white solid.
Example 4
General Procedure for the Preparation Propargyl Ether 43 Via the
Mitsunobu Reaction
[0527] Exemplified for 1-[(1-methyl-2-propyn-1-yl)oxy]naphthalene
(43, R.sup.1=Me, X=Y=CH): To a solution of 1-naphthol (200 mg, 1.38
mmol) and but-3-yn-2-ol (146 mg, 2.07 mmol) in anhydrous DCM (10
mL) under a N.sub.2 atmosphere and at 0.degree. C. was added
Ph.sub.3P (435 mg, 1.66 mmol) portion wise. The reaction mixture
was stirred for 10 min and then DEAD (360 mg, 2.07 mmol) (70%
solution in toluene) was slowly added. The resulting reaction
solution was stirred at the room temperature for 24 h and then
diluted with water (50 mL) and extracted with chloroform
(3.times.50 mL). The combined organic extracts were washed with
brine, dried over anhydrous MgSO.sub.4, filtered concentrated in
vacuo and the residue was purified by flash column chromatography
using ethyl acetate/n-hexane (a gradient of 5-10%) to furnish
1-[(1-methyl-2-propyn-1-yl)oxy]naphthalene (176 mg, 65%) as a
viscous oil.
Example 5
General Procedure for the Preparation Propargyl Ether 43 Via the
Corey-Fuchs Reaction
[0528] Exemplified for the synthesis of
1-(1-methylethyl)-2-propyn-1-yl]oxy]naphthalene (43, R.sup.1=i-Pr,
X=Y=CH): A the solution of 41 (R.sup.1=i-Pr, R.sup.2=CO.sub.2OH,
X=Y=CH, 880 mg, 3.27 mmol) in anhydrous THF was cooled to 0.degree.
C. and then LiAlH.sub.4 (311 mg, 8.18 mmol) was added. The reaction
mixture was stirred for 5 h at 0.degree. C. and then quenched with
water (50 mL). The mixture was stirred until the organic and
aqueous layers separated. The mixture was extracted with chloroform
(3.times.100 mL). The combined organic layers were washed with
brine, dried over anhydrous MgSO.sub.4, filtered, concentrated in
vacuo and purified by flash column chromatography using ethyl
acetate/n-hexane (a gradient of 2:1) to give alcohol 41
(R.sup.1=i-Pr, R.sup.2=CH.sub.2OH, X=Y=CH, 466 mg, 62%) as a thick
viscous oil.
[0529] A flame dried two neck round bottom flask containing oxalyl
chloride (370 .mu.L, 4.34 mmol) in anhydrous DCM was cooled at
-78.degree. C. under a nitrogen atmosphere. Next, anhydrous DMSO
(679 mg, 8.7 mmol) was added drop wise via a syringe. The resulting
solution was allowed to stir at -78.degree. C. for 10 min and then
alcohol 41 (R.sup.1=i-Pr, R.sup.2=CH.sub.2OH, X=Y=CH, 400 mg, 1.74
mmol) dissolved in anhydrous DCM was added gradually via a syringe.
The resulting reaction mixture was allowed to stir for 1 h at
-78.degree. C. and then quenched with triethylamine (1.95 mL, 13.9
mmol) before being allowed to warm to room temperature. The
reaction mixture was extracted with DCM (3.times.50 mL), washed
with brine, dried over anhydrous MgSO.sub.4, filtered, and
concentrated in vacuo to give aldehyde 42 (R.sup.1=i-Pr, X=Y=CH),
which was used without further purification.
[0530] A solution of 42 (R.sup.1=i-Pr, X=Y=CH, 360 mg, 1.58 mmol)
in DCM (10 mL) was cooled at 0.degree. C. and then Ph.sub.3P (1.24
gm, 4.74 mmol) and CBr.sub.4 (785 mg, 2.37 mmol) were sequentially
added. The resulting mixture was stirred at room temp for 2 h. The
reaction mixture was concentrated in vacuo and the Ph.sub.3PO was
precipitated by addition of n-hexane and removed by filtration. The
filtrate was concentrated and the residue purified using a filter
column (ethyl acetate/n-hexane as eluent). The resulting material
(360 mg, 1.58 mmol) was dissolved in anhydrous THF and cooled to
-78.degree. C. Next, n-BuLi (121 mg, 1.90 mmol) was gradually added
and the resulting solution stirred for 2 h at -78.degree. C. The
mixture was quenched with water (50 mL), allowed to stir at room
temperature for 30 min, and then extracted with ethyl acetate
(3.times.100 mL). The combined organic extracts were dried over
MgSO.sub.4, filtered, concentrated in vacuo and purified by flash
column chromatography eluting with ethyl acetate/n-hexane to
furnish 1-(1-methylethyl)-2-propyn-1-yl]oxy]naphthalene (43,
R.sup.1=i-Pr, X=Y=CH, 282 mg, 72%) as a white solid.
Example 6
General Procedure for the Preparation of 1-H-1,2,3-triazoles 44
(Examples 6-24)
[0531] Exemplified for
1-(4-chlorophenyl)-4-(2-methyl-1-(1-naphthalenyloxy)propyl)-1H-1,2,3-tria-
zole (3): A single neck round bottom flask under an argon
atmosphere was charged with 16 (R.sup.1=i-Pr, X=Y=CH, 114 mg, 0.51
mmol), anhydrous acetonitrile (5 mL), 1-azido-4-chlorobenzene (78.3
mg, 0.51 mmol) and DIPEA (254 .mu.L, 1.53 mmol). The reaction
mixture was allowed to stir at room temperature for 10 min and then
finely powdered CuI (194.2 mg, 1.02 mmol) was added portion wise.
After 30 min of stirring at room temperature, the reaction mixture
was quenched with saturated aqueous NH.sub.4Cl, diluted with water
(50 mL) and extracted with chloroform (3.times.50 mL). The combined
organic extracts were washed with brine, dried over anhydrous
MgSO.sub.4, filtered, concentrated in vacuo and purified by flash
column chromatography using ethyl acetate/n-hexane (a gradient of
10-20%) to furnish 3 (167 mg, 87%) as a gelatinous solid. .sup.1H
NMR (CDCl.sub.3, 400 MHz) .delta. 1.16, 1.20 (dd, J=6.5, 22.0 Hz,
6H), 2.47-2.53 (m, 1H), 5.55 (d, J=5.0 Hz, 1H), 6.80 (d, J=8.0 Hz,
1H), 7.25 (m, 1H), 7.37-7.43 (m, 3H), 7.49 (m, 2H), 7.61 (d, J=9.0
Hz, 2H), 7.79 (m, 2H), 8.38 (m, 1H); ESI-HRMS for
C.sub.22H.sub.21ClN.sub.3O(M+H).sup.+calcd. 378.1373. Found
378.1383.
Example 7
1-(4-chlorophenyl)-4-(1-(naphthalene-1-yloxy)ethyl)-1H-1,2,3-triazole
(1)
[0532] mp 98-100.degree. C.; .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 1.91 (d, J=6.0 Hz, 3H) 5.90 (q, J=6.8, 12.8 Hz, 1H), 6.92
(d, J=7.2 Hz, 1H), 7.26 (s, 1H), 7.31 (t, J=8.8 Hz, 1H), 7.41-7.51
(m, 5H), 7.63 (d, J=8.0 Hz, 2H), 7.80 (m, 1H), 7.88 (s, 1H), 8.34
(m, 1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 22.56, 69.90,
107.02, 119.08, 120.98, 121.87, 122.19, 125.53, 126.07, 126.14,
126.66, 127.80, 130.07, 134.73, 134.82, 135.64, 151.38, 153.21;
ESI-HRMS for C.sub.20H.sub.17ClN.sub.3O (M+H).sup.+ calcd.
350.1060. Found 350.1074.
Example 8
1-(2,6-dichlorophenyl)-4-(1-(naphthalen-1-yloxy)ethyl)-1H-1,2,3-triazole
(6)
[0533] yield 86%; Gummy solid; .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta. 1.98 (d, J=6.0 Hz, 3H), 5.92 (q, J=6.5, 13.0 Hz, 1H), 6.91
(d, J=8.0 Hz, 1H), 7.26 (s, 1H), 7.31 (t, J=8.0 Hz, 1H), 7.37-7.49
(m, 6H), 7.79 (m, 1H) 8.32 (m, 1H); ESI-HRMS for
C.sub.20H.sub.16Cl.sub.2N.sub.3O (M+H).sup.+calcd. 384.0670. Found
384.0672.
Example 9
4-(1-(4-chloronaphthalen-1-yloxy)ethyl)-1-(4-chlorophenyl)-1H-1,2,3-triazo-
le (7) yield 84%; Gummy solid; .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta. 1.91 (d, J=6.5 Hz, 3H), 5.86 (q, J=6.5, 13.5 Hz, 1H), 6.85
(d, J=8.0 Hz, 1H), 7.26 (s, 1H), 7.39 (d, J=8.5 Hz, 1H), 7.46 (d,
J=8.5 Hz, 1H), 7.56 (t, J=8.5 Hz, 1H), 7.62 (t, J=8.0 Hz, 3H), 7.87
(s, 1H), 8.20 (d, J=8.5 Hz, 1H), 8.36 (d, J=8.5 Hz, 1H); ESI-HRMS
for C.sub.20H.sub.16Cl.sub.2N.sub.3O (M+H).sup.+calcd. 384.0670.
Found 384.0684.
Example 10
4-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
(13) yield 91%; mp. 128-130.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.95 (d, J=6.4 Hz, 3H), 5.97 (q, J=6.4, 12.8 Hz,
1H), 6.89 (d, J=5.2 Hz, 1H), 7.47 (d, J=8.8 Hz, 2H), 7.53 (t, J=8.0
Hz, 1H), 7.64 (d, J=8.8 Hz, 2H), 7.71 (t, J=7.2 Hz, 1H), 7.91 (s,
1H), 8.03 (d, J=8.4 Hz, 1H), 8.28 (d, J=7.6 Hz, 1H), 8.70 (d, J=4.4
Hz, 1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 22.21, 70.05,
102.42, 119.17, 121.92, 121.99, 125.93, 129.23, 130.06, 130.14,
134.98, 135.48, 149.59, 150.01, 151.51, 160.17; ESI-HRMS for
C.sub.19H.sub.16ClN.sub.4O (M+H).sup.+calcd. 351.1013. Found
351.1002.
Example 11
4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
(14)
[0534] yield 89%; mp. 62-64.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.95 (d, J=6.4 Hz, 3H), 5.97 (q, J=6.4, 12.8 Hz,
1H), 6.87 (d, J=5.6 Hz, 1H), 7.26 (s, 1H), 7.53 (t, J=7.6 Hz, 1H),
7.57 (s, 2H), 7.71 (t, J=7.2 Hz, 1H), 7.85 (s, 1H), 7.91 (s, 1H),
8.03 (d, J=8.4 Hz, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.70 (d, J=4.4 Hz,
1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 22.21, 69.96,
102.37, 119.13, 119.69, 121.72, 121.97, 122.50, 125.98, 129.26,
130.10, 131.68, 133.35, 134.23, 135.98, 149.61, 150.28, 151.50,
160.10; Anal. (C.sub.19H.sub.14Cl.sub.2N.sub.4O) C, H, N.
Example 12
4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzonitrile
(15)
[0535] yield 87%; mp. 176-178.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.96 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.0, 12.8 Hz,
1H), 6.87 (d, J=4.8 Hz, 1H), 7.26 (s, 1H), 7.54 (t, J=7.2 Hz, 1H),
7.72 (t, J=7.2 Hz, 1H), 7.80 (d, J=8.8 Hz, 2H), 7.88 (d, J=8.0 Hz,
2H), 8.03 (m, 1H), 8.28 (d, J=8.0 Hz, 1H), 8.70 (s, 1H); .sup.13C
NMR (CDCl.sub.3, 100 MHz) .delta. 22.16, 69.90, 102.35, 112.86,
117.78, 118.98, 120.82, 121.94, 126.02, 129.25, 130.15, 134.11,
139.74, 149.59, 150.55, 151.45, 160.07; ESI-HRMS for
C.sub.20H.sub.16N.sub.5O (M+H).sup.+calcd. 342.1355. Found
342.1355.
Example 13
2-chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzonitri-
le (16)
[0536] yield 85%; mp. 194-196.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.96 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.0, 12.8 Hz,
1H), 6.84 (d, J=4.8 Hz, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.72 (t, J=8.0
Hz, 1H), 7.79 (q, J=8.4, 15.2 Hz, 2H), 7.97 (s, 1H), 8.00 (s, 1H),
8.04 (d, J=8.8 Hz, 1H), 8.23 (d, J=8.0 Hz, 1H), 8.69 (d, J=4.8 Hz,
1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 22.14, 69.81,
102.29, 113.53, 115.12, 118.60, 119.02, 121.51, 121.65, 121.91,
126.06, 129.27, 130.18, 135.53, 138.93, 140.24, 149.60, 150.82,
151.43, 159.98; ESI-HRMS for C.sub.20H.sub.15ClN.sub.5O (M+H).sup.+
calcd. 376.0965. Found 376.0975.
Example 14
(R)-5-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
(21)
[0537] yield 82%; mp. 94-96.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.92 (d, J=6.4 Hz, 3H), 5.89 (q, J=6.0, 12.8 Hz,
1H), 7.00 (d, J=8.0 Hz, 1H), 7.40 (dd, J=3.6, 8.0 Hz, 1H), 7.46 (d,
J=8.4 Hz, 2H), 7.55 (t, J=8.4 Hz, 1H), 7.65 (d, J=8.0 Hz, 2H), 7.69
(d, J=8.8 Hz, 1H), 7.88 (s, 1H), 8.65 (d, J=8.0 Hz, 1H), 8.91 (s,
1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 22.36, 70.17,
107.39, 119.10, 120.52, 121.40, 121.87, 122.32, 129.56, 130.10,
131.00, 134.85, 135.56, 149.36, 150.76, 150.95, 152.95; ESI-HRMS
for C.sub.19H.sub.16N.sub.4OCl (M+H).sup.+ calcd. 351.1013. Found
351.1002.
Example 15
(R)-4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
(28) yield 91%; mp. 62-64.degree. C.; .sup.1H NMR (CDCl.sub.3, 400
MHz) .delta. 1.95 (d, J=6.8 Hz, 3H), 5.97 (q, J=6.4, 12.8 Hz, 1H),
6.87 (d, J=5.2 Hz, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.58 (s, 2H), 7.72
(t, J=7.2 Hz, 1H), 7.85 (s, 1H), 7.91 (s, 1H), 8.05 (d, J=8.8 Hz,
1H), 8.28 (d, J=8.4 Hz, 1H), 8.70 (bs, 1H); ESI-HRMS for
C.sub.19H.sub.15N.sub.4OCl.sub.2(M+H).sup.+ calcd. 385.0623. Found
385.0605.
Example 16
(S)-4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
(30)
[0538] yield 89%; mp. 64-66.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.95 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.8, 12.8 Hz,
1H), 6.88 (d, J=4.8 Hz, 1H), 7.54 (t, J=6.4 Hz, 1H), 7.58 (d, J=1.2
Hz, 2H), 7.72 (m, 1H), 7.85 (m, 1H), 7.91 (s, 1H), 8.04 (d, J=8.0
Hz, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.70 (d, J=5.6 Hz, 1H); ESI-HRMS
for C.sub.19H.sub.15N.sub.4OCl.sub.2(M+H).sup.+Calcd. 385.0623.
Found 385.0628.
Example 17
(R)-2-chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzon-
itrile (29)
[0539] yield 92%; mp. 176-178.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.96 (d, J=6.0 Hz, 3H), 5.98 (q, J=6.0, 12.8 Hz,
1H), 6.84 (d, J=5.6 Hz, 1H) 7.55 (t, J=7.2 Hz, 1H), 7.71-7.83 (m,
3H), 7.97 (m, 2H), 8.04 (d, J=8.4 Hz, 1H), 8.28 (d, J=8.8 Hz, 1H),
8.70 (d, J=5.2 Hz, 1H); ESI-HRMS for C.sub.20H.sub.15N.sub.5OCl
(M+H).sup.+calcd. 376.0965. Found 376.0964.
Example 18
(S)-2-chloro-4-(4-(1-(quinolin-4-yloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzon-
itrile (31)
[0540] yield 91%; mp. 176-178.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.96 (d, J=6.8 Hz, 3H), 5.98 (q, J=6.0, 12.8 Hz,
1H), 6.84 (d, J=5.6 Hz, 1H), 7.54 (t, J=8.0 Hz, 1H), 7.71-7.82 (m,
3H), 7.98 (m, 2H), 8.04 (d, J=8.0 Hz, 1H), 8.28 (d, J=8.4 Hz, 1H),
8.70 (d, J=4.8 Hz, 1H); ESI-HRMS for C.sub.20H.sub.15N.sub.5OCl
(M+H).sup.+calcd. 376.0965. found 376.0974.
Example 19
4-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)quinoline
(17)
[0541] yield 89%; mp. 230-232.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 5.50 (s, 2H), 6.34 (d, J=8.0 Hz, 1H), 7.39 (t,
J=7.2 Hz, 1H), 7.47 (d, J=8.4 Hz, 2H), 7.57-7.65 (m, 4H), 7.74 (d,
J=8.0 Hz, 1H), 7.77 (s, 1H), 8.46 (d, J=8.0 Hz, 1H); .sup.13C NMR
(CDCl.sub.3, 100 MHz) .delta. 48.81, 111.20, 115.77, 120.21,
121.92, 124.28, 127.49, 127.58, 130.24, 132.75, 135.29, 139.87,
143.11, 143.93, 178.46; ESI-HRMS for C.sub.18H.sub.14ClN.sub.4O
(M+H).sup.+calcd. 337.0856. Found 337.0847.
Example 20
5-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
1-oxide (25) yield 81%; mp. 165-167.degree. C.; .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 1.93 (d, J=6.0 Hz, 3H), 5.90 (q,
J=6.812.8 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H) 7.26 (t, J=6.0 Hz, 1H),
7.47 (d, J=8.4 Hz, 2H), 7.60 (t, J=8.4 Hz, 1H), 7.65 (d, J=8.4 Hz,
2H), 7.91 (s, 1H), 8.20 (d, J=8.4 Hz, 1H) 8.29 (d, J=9.2 Hz, 1H),
8.53 (d, J=5.2 Hz, 1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta.
22.27, 70.58, 109.66, 112.34, 119.20, 120.13, 121.03, 121.91,
123.94, 130.26, 130.73, 135.01, 135.49, 136.33, 142.76, 150.15,
153.56; ESI-HRMS for C.sub.19H.sub.16N.sub.4O.sub.2Cl
(M+H).sup.+calcd. 367.0962. Found 367.0977.
Example 21
(R)-5-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
1-oxide (32)
[0542] yield 88%; mp. 184-186.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.93 (d, J=6.0 Hz, 3H), 5.90 (q, J=6.4, 12.8 Hz,
1H), 7.14 (d, J=8.0 Hz, 1H), 7.28 (m, 3H), 7.48 (d, J=9.2 Hz, 2H),
7.61 (t, J=8.4 Hz, 1H), 7.65 (d, J=9.2 Hz, 2H), 7.91 (s, 1H), 8.21
(d, J=8.4 Hz, 1H), 8.30 (d, J=9.2 Hz, 1H), 8.53 (d, J=5.2 Hz, 1H);
ESI-HRMS for C.sub.19H.sub.16N.sub.4O.sub.2Cl (M+H).sup.+calcd.
367.0962. Found 367.0947.
Example 22
4-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
1-oxide (23)
[0543] yield 88%; mp. 158-160.degree. C.; .sup.1H NMR
(d.sub.6-DMSO, 400 MHz) .delta. 1.85 (d, J=6.8 Hz, 3H), 6.09 (q,
J=6.0, 12.8 Hz, 1H), 7.20 (d, J=6.8 Hz, 1H), 7.67 (d, J=8.4 Hz,
2H), 7.74 (t, J=7.6 Hz, 1H), 7.86 (t, J=8.4 Hz, 1H), 7.95 (d, J=9.2
Hz, 2H), 8.25 (d, J=8.8 Hz, 1H), 8.50 (dd, J=6.4, 12.0 Hz, 2H),
9.06 (s, 1H); .sup.13C NMR (d.sub.6-DMSO, 100 MHz) .delta. 20.58,
69.53, 103.30, 119.27, 121.56, 121.82, 122.67, 122.80, 128.14,
129.87, 130.79, 133.06, 135.32, 135.48, 140.72, 148.38, 150.38;
ESI-HRMS for C.sub.19H.sub.16N.sub.4O.sub.2Cl (M+H).sup.+calcd.
367.0962. found 367.0948.
Example 23
4-(1-(1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
1-oxide (26) yield 92%; mp. 216-218.degree. C.; .sup.1H NMR
(d.sub.6-DMSO, 400 MHz) .delta. 1.85 (d, J=6.0 Hz, 3H), 6.09 (q,
J=6.0, 12.8 Hz, 1H), 7.19 (d, J=6.4 Hz, 1H), 7.74 (t, J=8.0 Hz,
1H), 7.86 (m, 2H), 7.98 (d, J=9.2 Hz, 1H), 8.26 (m, 2H), 8.50 (dd,
J=7.2, 12.0 Hz, 2H), 9.12 (s, 1H); .sup.13C NMR (d.sub.6-DMSO, 100
MHz) .delta. 20.58, 69.47, 103.33, 119.28, 120.16, 121.74, 121.85,
122.67, 122.78, 128.15, 130.76, 131.09, 131.81, 132.33, 135.39,
136.03, 140.71, 148.53, 150.21; ESI-HRMS for
C.sub.19H.sub.15N.sub.4O.sub.2Cl.sub.2(M+H).sup.+calcd. 401.0571.
Found 401.0566.
Example 24
(R)-4-(1-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)ethoxy)quinoline
1-oxide (33)
[0544] yield 91%; mp. 172-174.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.96 (d, J=8.8 Hz, 3H), 5.92 (q, J=6.0, 12.8 Hz,
1H), 6.88 (d, J=6.8 Hz, 1H), 7.48 (d, J=8.8 Hz, 2H), 7.66 (d, J=8.8
Hz, 3H), 7.80 (t, J=7.2 Hz, 1H), 7.96 (s, 1H), 8.28 (d, J=8.0 Hz,
1H), 8.39 (d, J=6.8 Hz, 1H), 8.73 (d, J=8.4 Hz, 1H); ESI-HRMS for
C.sub.19H.sub.16N.sub.4O.sub.2Cl (M+H).sup.+calcd. 367.0962. Found
367.0948.
Example 25
General Procedure for the Preparation Propargyl Ether Quinoline
N-Oxides
[0545] Exemplified for (R)-5-(but-3-yn-2-yloxy)quinoline 1-oxide
(43, R.sup.1=(R)-Me, X=N.sup.+--O.sup.-, Y-CH): To a 0.degree. C.
solution of 43 (R.sup.1=(R)-Me, X=N, Y=CH, 120 mg, 0.61 mmol) in
anhydrous dichloromethane under a nitrogen atmosphere was added
m-chloroperbenzoic acid (163 mg, 0.73 mmol, 77%). The reaction
mixture was stirred at room temperature for 2 h, concentrated in
vacuo and purified by flash column chromatography eluting with
methanol/chloroform (a gradient of 5-10%) to furnish
(R)-5-(but-3-yn-2-yloxy)quinoline 1-oxide (43, R.sup.1=(R)-Me,
X=N.sup.+--O.sup.-, Y=CH, 120 mg, 93%) as a white solid. mp.
156-158.degree. C.; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 1.82
(d, J=6.8 Hz, 3H), 2.54 (s, 1H), 5.05 (q, J=6.8, 13.6 Hz, 1H), 7.20
(d, 1H, J=8.0 Hz, 1H) 7.26 (t, J=8.0 Hz, 3H), 7.68 (t, J=9.2 Hz,
1H), 8.15 (d, J=9.2 Hz, 1H), 8.35 (d, J=8.8 Hz, 1H), 8.54 (d, J=5.2
Hz, 1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 22.32, 64.68,
75.09, 81.96, 109.52, 112.49, 120.09, 121.28, 123.85, 130.57,
136.32, 142.64, 153.36; ESI-HRMS for
C.sub.13H.sub.12NO.sub.2(M+H).sup.+calcd. 214.0868. Found
214.0875.
Example 26
Other 1,2,3-Triazoles were Prepared According to FIG. 9
Example 27
General Synthetic Strategy Towards Various Amides
[0546] Various amide analogs were prepared according to FIG. 35.
Ethyl glyoxylate was allowed to react with c-PrMgBr to give a
corresponding alcohol that was subsequently converted to bromide 46
(R=c-Pr) with carbon tetrabromide and triphenylphosphine. Various
other bromide derivatives of 46 were commercially available.
Treatment of 46 with 1-naphthol in the presence of base
(K.sub.2CO.sub.3) gave ester 41. The ester was saponified with 3 N
sodium hydroxide in THF to give acid 41, which was subsequently
converted to amide 188 (X=CH) with the aid of EDCI.HCl. In the case
of a quinoline analog of 188 (X=N), the acetyl chloride derivative
186 was first converted to amide 187, which was treated with
4-hydroxyquinoline to give 188 (X=N).
Example 28
General Procedure for the Synthesis of
N-(4-chlorophenyl)-2-(1-naphthalenyloxy)acetamides (188, X=CH)
(Examples 29-31)
[0547] Exemplified for
N-(4-chlorophenyl)-2-cyclopropyl-2-(1-naphthalenyloxy)acetamide
(119): To a solution of 2-cyclopropyl-2-(1-naphthalenyloxy)acetic
acid (120 mg, 0.49 mmol) and 4-chloroaniline (44.0 .mu.L, 0.49
mmol) in anhydrous DCM (10 mL) under N.sub.2 cooled at 0.degree. C.
was added EDCI-HCl (187.9 mg, 0.98 mmol) portion wise. The
resulting solution was stirred at room temperature for 12 h. The
reaction mixture was diluted with water (50 mL) and extracted with
ethyl acetate (3.times.100 mL). The organic extracts were combined,
washed with brine, dried over anhydrous MgSO.sub.4, filtered, and
concentrated in vacuo. The residue was purified by flash column
chromatography using ethyl acetate/n-hexane (a gradient of 5-10%)
to furnish 119 (148 mg, 86%) as a white solid. mp 204-206.degree.
C.; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 0.65-0.78 (m, 4H0,
1.52 (m, 1H), 4.41 (d, J=6.4 Hz, 1H), 6.70 (d, J=8.0 Hz, 1H), 7.25
(d, J=6.0 Hz, 3H), 7.43 (d, J=8.4 Hz, 2H), 7.61-7.70 (m, 3H), 7.97
(s, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.34 (d, J=8.0 Hz, 1H); .sup.13C
NMR (CDCl.sub.3, 100 MHz) .delta. 2.70, 3.16, 14.42, 82.72, 108.01,
115.68, 121.47, 122.03, 122.96, 127.64, 128.43, 129.28, 129.63,
130.07, 132.96, 135.61, 152.83, 169.27; ESI-HRMS for
C.sub.21H.sub.17ClNO.sub.2 (M-H).sup.+calcd. 350.0948. Found
350.0956.
Example 29
N-(4-chlorophenyl)-2-(1-naphthalenyloxy)propanamide (93)
[0548] mp. 146-148.degree. C.; .sup.1H NMR (CDCl.sub.3, 400 MHz):
.delta. 1.79 (d, J=6.8 Hz, 3H), 4.98 (q, J=6.8, 13.2 Hz, 1H), 6.87
(d, J=7.6 Hz, 1H), 7.26 (d, J=8.4 Hz, 2H), 7.37 (t, J=8.4 Hz, 1H),
7.46 (d, J=8.8 Hz, 2H), 7.56 (dd, J=7.8, 12.5 Hz, 3H), 7.86 (m,
1H), 8.24-8.32 (m, 2H). .sup.13C NMR (CDCl.sub.3, 100 MHz): .delta.
19.08, 76.26, 107.41, 121.42, 121.54, 122.47, 125.91, 126.04,
126.16, 127.03, 128.14, 129.26, 129.95, 134.93, 135.75, 152.61,
170.58; Anal. (C.sub.19H.sub.16ClNO.sub.2) C, H, N.
Example 30
N-(4-chlorophenyl)-.alpha.-(1-naphthalenyloxy)benzeneacetamide
(102)
[0549] yield 92%; mp. 176-178.degree. C.; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 5.86 (s, 1H), 6.88 (d, J=8.0 Hz, 1H), 7.26-7.61
(m, 11H), 7.68 (d, J=7.2 Hz, 2H), 7.87 (d, J=7.2 Hz, 1H), 7.37 (d,
J=8.0 Hz, 1H), 8.44 (s, 1H). .sup.13C NMR (CDCl.sub.3, 100 MHz):
.delta. 81.09, 107.87, 121.41, 121.47, 122.58, 125.66, 126.01,
126.27, 126.61, 127.02, 128.25, 129.12, 129.15, 129.27, 130.06,
134.94, 135.71, 135.98, 152.34, 168.11; Anal.
(C.sub.24H.sub.18ClNO.sub.2) C, H, N.
Example 31
N-(4-chlorophenyl)-3-methyl-2-(1-naphthalenyloxy)butanamide
(116)
[0550] yield 91%; mp. 150-152.degree. C., .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.22 (dd, J=6.8, 22.8 Hz, 6H), 2.54 (m, 1H), 4.68
(d, J=4.4 Hz, 1H), 6.83 (d, J=8.0 Hz, 1H), 7.25 (m, 2H), 7.35 (t,
J=8.0 Hz, 1H), 7.40 (d, J=8.8 Hz, 2H), 7.52 (d, J=8.0 Hz, 1H),
7.54-7.60 (m, 2H), 7.86 (m, 1H), 8.01 (s, 1H), 8.38 (m, 1H);
.sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 17.47, 19.54, 32.48,
84.51, 106.81, 121.53, 122.20, 125.71, 126.12, 127.04, 128.15,
129.21, 129.97, 134.92, 135.57, 153.52, 169.68; Anal.
(C.sub.21H.sub.20ClNO.sub.2) C, H, N.
Example 32
Synthesis of 2-bromo-N-(4-chlorophenyl)propanamide (187)
[0551] To a solution of 4-chloroaniline (400 mg, 3.13 mmol) in
anhydrous dichloromethane at room temperature under N.sub.2 was
added slowly 2-bromopropanoyl chloride (474 .mu.L, 4.7 mmol). The
reaction mixture was stirred at room temperature for 2 h and then
diluted with water (50 mL) and extracted with ethyl acetate
(3.times.50 mL). The organic extracts were combined, washed with
brine, dried over anhydrous MgSO.sub.4 and concentrated in vacuo to
give 2-bromo-N-(4-chlorophenyl)propanamide, which was used without
further purifications.
Example 33
Synthesis N-(4-chlorophenyl)-2-[[4-quinolinyl]oxy]propanamide
(114)
[0552] To a solution of 4-hydroxyquinoline (100 mg, 0.69 mmol) in
anhydrous DMF under N.sub.2 was added K.sub.2 CO.sub.3 (286 mg,
2.10 mmol) and a solution of 2-bromo-N-(4-chlorophenyl)propanamide
(218 mg, 0.83 mmol) in DMF. The reaction mixture was stirred for 12
h at room temperature before being diluted with water (50 mL) and
extracted with chloroform (3.times.50 mL). The combined organic
extracts were dried over anhydrous MgSO.sub.4, filtered,
concentrated in vacuo and purified by flash chromatography using
ethyl acetate/n-hexane (a gradient of 10%) to furnish 114 (207 mg,
92% yield) as a white solid. mp. 170-172.degree. C., .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 1.83 (d, J=6.4 Hz, 3H), 5.06 (q,
J=6.8, 13.6 Hz, 1H), 6.76 (d, J=5.6 Hz, 1H), 7.28 (d, J=8.4 Hz,
2H), 7.46 (d, J=8.4 Hz, 2H), 7.61 (t, J=8.0 Hz, 1H), 7.77 (t, J=7.6
Hz, 1H), 8.09 (d, J=7.2 Hz, 1H), 8.26 (d, J=8.4 Hz, 1H), 8.76 (d,
J=5.6 Hz, 1H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 18.72,
75.83, 102.05, 122.27, 121.32, 121.58, 126.61, 129.34, 129.60,
130.34, 130.51, 135.40, 149.74, 151.48, 159.47, 169.20; ESI-HRMS
for C.sub.18H.sub.16N.sub.2O.sub.2Cl (M+H).sup.+calcd. 327.0900.
found 327.0901.
Example 34
C. parvum IMPDH Screen
[0553] Recombinant C. parvum IMPDH was expressed in bacteria and
purified as described previously [N. N. Umejiego et al, J Biol Chem
279, 40320-40327 (2004)].
[0554] Determining the IC.sub.50 values. Inhibitors at varying
concentrations (25 .mu.M-5 .mu.M) were incubated with 10 nM C.
parvum in assay buffer for 10 min at room temperature. The reaction
was initiated by the addition of NAD and IMP for final
concentrations of 300 .mu.M and 150 .mu.m, respectively.
Selectivity was measured against human type II and T. foetus IMPDH
at 25.degree. C. in assay buffer. The former was assayed in the
presence of 300 .mu.M NAD.sup.+, 40 .mu.M IMP and 160 nM human type
II IMPDH, and the latter in the presence of 300 .mu.M NAD.sup.+, 20
.mu.M IMP and 28 nM T. foetus IMPDH. The production of NADH was
monitored spectrophotometrically at 340 nm (=6.22 mM.sup.-1
cm.sup.-1) using a Hitachi U-2000 spectrophotometer.
[0555] IC.sub.50 values were calculated for each inhibitor
according to the following equation:
.upsilon..sub.i=.upsilon..sub.o/(1+[I]/IC.sub.50), using the
SigmaPlot program (SPSS, Inc.).
Example 35
Heliobacter pylori, Borrelia burgdorferi, and Streptococcus
pyogenes IMPDH screen
[0556] The IC.sub.50 values of various compounds of the invention
were determined for recombinant IMPDHs from H. pylori, B.
burgdorferi, and S. pyogenes.
Example 36
Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase
Inhibitor Selectivity
[0557] Cryptosporidium parvum is a potential bio-warfare agent, an
important AIDS pathogen and a major cause of diarrhea and
malnutrition. No vaccines or effective drug treatment exist to
combat Cryptosporidium infection. This parasite relies on inosine
5'-monophosphate dehydrogenase (IMPDH) to obtain guanine
nucleotides and inhibition of this enzyme blocks parasite
proliferation. Here we report the first crystal structures of
CpIMPDH. These structures reveal the structural basis of inhibitor
selectivity and suggest a strategy for further optimization. Using
this information, we have synthesized low nanomolar inhibitors that
display 10.sup.3 selectivity for the parasite enzyme over human
IMPDH2.
[0558] Cryptosporidium spp. are a major cause of the "vicious
cycle" of diarrhea and malnutrition in the developing world and a
potential bioterrorism agent. This disease is prolonged and
life-threatening in immuno-compromised patients. Currently no
effective therapy exists for Cryptosporidium infections. The
parasite obtains guanine nucleotides via a streamlined pathway that
requires inosine 5'-monophosphate dehydrogenase (IMPDH). Curiously,
the gene encoding CpIMPDH appears to have been obtained from a
bacteria via lateral gene transfer; we have exploited this
unexpected divergence of parasite and host enzymes to identify
CpIMPDH-specific inhibitors in a high throughput screen. Here we
report x-ray crystal structures of CpIMPDH that explain the
selectivity of one inhibitor series and use this information to
design more potent and selective analogs.
[0559] Recombinant CpIMPDH was purified as described previously and
crystallized using the hanging drop vapor diffusion method. Protein
solution (4 mg/mL IMPDH, 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 5%
glycerol and 2 mM DTT) was mixed with well solution (34% PEG 4000,
25 mM sodium acetate and 100 mM Tris-HCl, pH 8.5) in a 1:1 ratio.
Data were collected from a single crystal at 100K at beamline 8-BM
at Advanced Photon Source (Argonne National Laboratory, Argonne,
Ill.). The crystals had the symmetry of space group
P2.sub.12.sub.12. The asymmetric unit contains one tetramer, which
is the active form of IMPDH. The structure was solved to 3.2 .ANG.
resolution (R=27%, R.sub.free=33%) by molecular replacement using
the structure of IMPDH from Borrelia burgdorferi (PDB accession
LEEP) as a search model. Only 301 of 400 residues are visible in
the most structured monomer; the disordered regions include
catalytically important segments 214-222, 299-333 and 380-400 as
well as residues 92-122, which are not required for enzymatic
activity. Unfortunately, we were unable to improve this crystal
form. This structure has been deposited in the PDB (3FFS).
[0560] To facilitate crystallization, residues 90-134 were replaced
with SerGlyGly; this modification has no effect on enzymatic
activity. A crystallization screen was performed in the presence of
IMP and various inhibitors that emerged from initial evaluation of
the SAR. Compound C64 (aka 174) was a particularly attractive
candidate for crystallization because of its improved potency
relative to the parent compound C (aka 123) and the presence of a
bromine atom which would allow the two aromatic groups to be easily
distinguished. Crystals were obtained in the presence of saturating
concentrations of inhibitor C64 (20 .mu.M), IMP (1 mM), 100 mM
sodium acetate, pH 4.6, 20 mM CaCl.sub.2 and 30% MPD under oil.
These crystals had the symmetry of space group P2.sub.1 with two
tetramers in the asymmetric unit. Data were collected at a
wavelength of 0.9194 .ANG., enabling the simultaneous collection of
bromine k-edge anomalous dispersion data. The structure was solved
by molecular replacement to 2.8 .ANG. resolution using the native
CpIMPDH structure as the starting model (R=22.4%,
R.sub.free=26.6%).
[0561] While the overall structure of the E.cndot.IMP.cndot.C64
complex is similar to that of the unliganded enzyme, several
additional residues are observed. Residues 214-226, which include
the catalytic Cys219, are observed in most of the monomers, as are
parts of the active site flap (residues 302-330) containing the
characteristic ArgTyr motif. Lastly, the SerGlyGly linker is
visible in all monomers. Electron density for IMP is observed in
all eight monomers. Monomers B, D and H contained extra electron
density near IMP (FIG. 70). Bromine k-edge anomalous dispersion
maps allowed the unambiguous assignment of the bromine atom in C64
in all three monomers. The rest of C64 was modeled into the
remaining electron density; similar conformations of C64 are
obtained in all three monomers. This structure has been deposited
in the PDB (3 KHJ).
[0562] Surprisingly, C64 binds in an unprecedented fashion.
Inhibitors of human IMPDH2 such as mycophenolic acid and
merimepodib bind in the nicotinamide subsite, stacking against the
purine ring of IMP in a parallel fashion, and extend either into
the NAD site or into a pocket adjacent the active site but within
the same monomer. In contrast, the thiazole ring of C64 stacks
against the purine ring of IMP perpendicularly, and the remainder
of C64 extends across the subunit interface into a pocket in the
adjacent monomer, where the bromoaniline moiety interacts with
Tyr358' (where ' denotes a residue from the adjacent subunit; FIG.
71). This residue forms a hydrogen bonding network involving
Glu329, Ser354, Thr221 and possibly the amide nitrogen of C64 (FIG.
71). Ser22', Pro26', Ala165, Gly357' form the remainder of the
inhibitor binding pocket. With the exception of Thr221, all of
these residues are different in human IMPDHs (FIG. 71). Thus these
interactions account for the selectivity of C64 for CpIMPDH over
human IMPDHs.
[0563] The structure also revealed the presence of a cavity
adjacent to the bromoaniline moiety (FIG. 70), which suggested that
more potent inhibitors might be created by increasing the bulk of
this substituent. Additional benzimidazole based inhibitors were
prepared by condensing o-phenylenediamine (FIG. 55, 1) with
thiazole carboxaldehydes (FIG. 55, 2) in the presence of the
oxidizing reagent sodium metabisulfite (FIG. 55, 3). The resulting
2-substituted benzimidazoles (FIG. 55, 4) were then coupled with
different bromoacetylamides (FIG. 55, 5) under mild basic
conditions to give the new analogs (FIG. 55, 6).
[0564] The CpIMPDH inhibitory activity of the compounds was
assessed by monitoring the production of NADH by fluorescence (FIG.
55). Replacing the p-MeO of the parent compound C with Cl or Br
increased potency by 10-fold (C10, aka 126) and 20-fold (C14, aka
130), respectively, as has been similarly observed with another
inhibitor series. To fill the cavity observed in the crystal
structure, the para-substituted aniline group was replaced with
3,4-dichloroaniline (C86) or 2-naphthylamine (C90); the addition of
a second Cl improved potency by a factor of 2, while fusing an
additional aromatic ring increased potency by a factor of 8.
Similar trends were observed when the thiazole ring was attached at
the 2-position (C61 aka 171, C64 aka 174, C84 and C90). None of the
compounds displayed significant inhibitory activity against human
IMPDH2. The best CpIMPDH inhibitor, C90, has an IC.sub.50=7.4 nM
with selectivity >10.sup.3 for the parasite enzyme.
[0565] In conclusion, the crystal structure of CpIMPDH reveals the
structural basis of inhibitor selectivity and a strategy for
further optimization. This information was used to design more
potent and selective inhibitors of CpIMPDH that are potential lead
compounds for the treatment of cryptosporidiosis.
Example 37
Structure-Activity Relationship Study of Selective
Benzimidazole-Based Inhibitors of C. parvum IMPDH
[0566] During a high throughput screening (HTS) process, the
benzimidazole analog C1 (compound 123) was identified (FIG. 21) as
a moderately potent but highly selective inhibitor for Cp-IMPDH
(IC.sub.50=1.2 .mu.M) with no detectable activity against the human
IMPDH-II enzyme (IC.sub.50>50 .mu.M). This molecule has
demonstrated uncompetitive inhibition with respect to IMP and
noncompetitive (mixed) inhibition with respect to NAD.sup.+. It was
also shown to bind the nicotinamide subsite and to directly or
indirectly impose on the ADP site. Herein, we report a
structure-activity relationship (SAR) study for this class of
inhibitors.
[0567] The benzimidazole analogs were synthesized following the
procedure outlined in FIG. 55. Various acetylamide derivatives
(FIG. 55, 3) were prepared by treating substituted anilines (FIG.
55, 1) with bromo acetylchloride (FIG. 55, 2) in dichloromethane
(DCM) and in the presence of catalytic amounts of
N,N-dimethylaminopyridine (DMAP). Various 2-substituted
benzimidazoles FIG. 55, 6) were prepared by condensing O-phenylene
diamine (FIG. 55, 4) with aromatic aldehydes followed by oxidation
in the presence of sodium metabisulfite. Finally, 2-substituted
benzimidazoles were coupled with the acetylamides (FIG. 55, 3) in
the presence of potassium carbonate to yield derivatives of C.
[0568] Evaluation of Cp-IMPDH inhibitory activity for the various
prepared compounds was conducted utilizing an assay measuring the
conversion of IMP to XMP by monitoring the production of NADH by
fluorescence emission in the presence of varying inhibitor
concentrations. Chlorine and bromine are found to be more effective
substituents at the para-position of the aniline ring (FIGS.
21-23). On the contrary, ortho and meta substitutions were devoid
of inhibition activity.
[0569] An electron donating group, such as thiomethyl (C39,
compound 154) at the para position. Interestingly increasing the
chain length, and making a benzyl derivative C18 results in the
loss of activity towards Cp-IMPDH. In addition, any branched
substitutions such as SO.sub.2Me (C40, compound 155) or isopropyl
(C43, compound 157) at the para position resulted the loss of
activity. Among all the derivatives we have synthesized, 2-napthyl
(C90) and 3,4-dichloro derivatives (C86) are found to be the most
potent inhibitors, which suggest that this part of the molecule
contributes to the steric factor for the binding process than
electronic factor.
[0570] However, surprisingly, the 1-naphthyl derivative C28
(compound 144) did not show any inhibition, whereas its positional
isomer, 2-napthyl derivative found to be a potent inhibitor, which
explains that indeed subtle changes in molecular orientation would
have significant influence in determining the binding ability.
Observed low IC.sub.50 of C90 (7 nM) suggesting that C90 molecule
could fit appropriately into the active site of the IMPDH. The SAR
of this part of the inhibitor is similar to that has been
previously reported with another series from our group. In
addition, we have investigated the role of active methylene group
by substituting various groups at the active methylene group, and
found that any subtle changes at that position led to lose of
inhibition ability (FIGS. 21-23).
[0571] Subsequently, the SAR of the thiazole ring, aniline ring is
fixed as 4-chloro derivative, 2, 3 and 4 thiazoles are found to be
twice as potent as the thiabendazole derivative C10 (compound 126)
(FIGS. 21-23). When the ring system is changed to thiophene, C62
(compound 172) showed high potency towards Cp-IMPDH inhibition with
20 nM IC.sub.50. Other heterocylic rings like pyrrole, oxazole, and
pyrazole are also well tolerated. Replacing the thiazole ring (C61,
compound 171) with thiophene (C62, compound 172) caused the drop in
IC.sub.50 values about 10 nM, however, replaced with pyrrole ring
(C65, compound 175) increased the IC.sub.50 values about 45 nM
compared to C61 (compound 171). Thus, compared to thiazole,
thiophene has effective while pyrrole proved to be much less
effective inhibitor. Oxazole derivative (C69) is not as potent as
the thiazole or thiophen derivatives but pyrazole derivative C100
was as potent as 2-thiazoles. When the thiazole ring is replaced by
a methyl group (C38, compound 153) the compound lost its activity
(FIGS. 21-23). Phenyl (C17, compound 133), pyridyl (C16, compound
132) rings are also tolerated in the 2-position. Substituted
phenyls (C31 (compound 147), C59 (compound 169)) are not as active
as the non-substituted.
[0572] Recently, the potent inhibitor C64 (compound 174) has been
co-crystallized with Cp-IMPDH. The co-crystallized structure of
Cp-IMPDH with C64 is solved and the SAR matches perfectly well with
the structure. 2-aromatic substitution in the benzimidazole portion
is important since it interacts with the IMP in the active site
according to the crystal structure. The amide bond is also very
important for the activity since it could potentially form hydrogen
bonding with the active site residues.
[0573] Bulkier substituents are better at the para position of the
aniline ring. Increasing the chain length either at the aniline as
well in the benzimidazole results in the loss of activity.
Replacing thiazole ring by phenyl, pyridyl and other heterocycles
retain the activity but replacement with methyl results in the loss
of activity. Replacing aniline by other non-aromatic cycles like
morpholine, piperidine results in the loss of activity.
[0574] Selectivity plays a major role in determining the success of
the drug molecules. After a detailed investigation of Cp-IMPDH
inhibition ability of C-series molecules, we have evaluated their
selectivity towards parasite Cp-IMPDH over human II-IMPDH.
Intriguingly, all the molecules were showed excellent selectivity
towards Cp-IMPDH over human II-IMPDH even though both the proteins
share high identity in their binding site.
[0575] In conclusion we have studied SAR of new series of
benzimidazole derivatives as potent inhibitors for Cp-IMPDH. Many
of the derivatives have shown nanomolar inhibition towards IMPDH.
The IC.sub.50 was improved to 2 nM (C91) from 1200 nM for the
identified lead compound C through a systematic SAR studies. Few of
the compounds showed increased efficacy with the cell based assays.
The crystal structure opens a new pathway to design structurally
diversified potent inhibitors for the selective inhibition of
Cp-IMPDH over Human II-IMPDH. The benzimidazole based CpIMPDH
inhibitors described herein could serve as lead compounds for drug
discovery to treat cryptosporidiosis.
[0576] Often in drug development, despite excellent inhibition
ability, several molecules fail to show significant stability under
metabolic conditions which is one of the key parameters for
defining potent drug molecules. Thus, metabolic stability of
selective inhibitors from several series has been investigated
using human and mouse microsomes and human and mouse plasma.
Results are summarized in FIG. 56. Several compounds showed
excellent stability with mouse microsomes and most compounds showed
excellent plasma stability over 2 hours, which is considered to be
significant stability.
Example 38
A Screening Pipeline for Antiparasitic Agents Targeting
Cryptosporidium IMPDH
[0577] Results
[0578] Engineering a Toxoplasma reporter parasite suitable for the
screening of CpIMPDH inhibitors. To facilitate screening of
antiparasitic activity, we constructed a T. gondii reporter
parasite that mirrors the Cryptosporidium purine metabolism. FIG.
57 summarizes the main differences between the two parasites in
this pathway. We started with a T. gondii knockout mutant that,
like C. parvum, lacks the ability to salvage xanthine and guanine
via HXGPRT (T. gondii-.DELTA.HXGPRT (10)) and introduced the
CpIMPDH gene under the control of a T. gondii promoter. Next, the
native T. gondii IMPDH gene was disrupted by replacing the entire
coding sequence with a chloramphenicol acetyl transferase cassette
using a new cosmid-based gene targeting approach. Successful
disruption of the gene was confirmed by PCR and Southern blotting;
note that numerous attempts by independent laboratories failed to
target this locus using smaller plasmid-based constructs. This
manipulation created strain T.
gondii-CpIMPDH-.DELTA.HXGPRT-.DELTA.TgIMPDH. Lastly, we introduced
a fluorescent protein cassette and isolated stable transgenic
parasites by cell sorting. The resulting strain is referred to as
T. gondii-CpIMPDH. The growth of this new parasite can be
conveniently monitored in live cultures in 96 or 384 well format
using a fluorescence plate reader. Similarly engineered fluorescent
versions of wild-type T. gondii and T. gondii-.DELTA.HXGPRT served
as controls.
[0579] Pharmacological validation of the Toxoplasma reporter
parasite. To determine if the proliferation of T. gondii-CpIMPDH
depends on CpIMPDH as designed, we performed fluorescence growth
assays in the presence of varying concentrations of mycophenolic
acid (MPA) comparing wild-type T. gondii, T. gondii-.DELTA.HXGPRT,
and T. gondii-CpIMPDH parasites (FIG. 57). MPA is a potent
inhibitor of eukaryotic IMPDHs including TgIMPDH but a very poor
inhibitor of prokaryotic IMPDHs. CpIMDPH is of prokaryotic origin
and not inhibited by MPA. As predicted, both wild-type T. gondii
and T. gondii-AHXGPRT are sensitive to MPA (FIG. 57), but T.
gondii-CpIMPDH is resistant (FIG. 57). T. gondii-AHXGPRT is the
most sensitive strain due to its inability to salvage
xanthine/guanine from the media (EC.sub.50=0.29 .mu.M versus 2.6
.mu.M for the wild-type strain; FIG. 63 details how an EC.sub.50
was derived from the parasite growth curves). Supplementation of
the media with xanthine (0.33 mM) essentially renders wild-type T.
gondii MPA resistant (EC.sub.50.gtoreq.78 .mu.M), but has no effect
on T. gondii-.DELTA.HXGPRT (FIG. 57). In contrast, the MPA
EC.sub.50>65 .mu.M for T. gondii-CpIMPDH is significantly
higher, as expected given the resistance of the prokaryotic
CpIMPDH, and is independent of xanthine supplementation (FIG.
57).
[0580] These results demonstrate that the T. gondii model system
provides a powerful tool for the evaluation of in vivo efficacy,
selectivity, and specificity of CpIMPDH inhibitors. Compounds that
selectively inhibit CpIMPDH will block the proliferation of T.
gondii-CpIMPDH but not the wild-type and T. gondii-AHXGPRT strains
that depend on an enzyme much like the human host. In contrast,
non-specific compounds that have off-target activities in the
parasite or the host cell will inhibit the growth of all three
strains. In the presence of xanthine, a general non-selective
inhibitor of both prokaryotic and eukaryotic IMPDH inhibitors will
block the proliferation of both T. gondii-CpIMPDH and T.
gondii-.DELTA.HXGPRT but will have no effect on the wild-type
strain; note that such compounds should be detected in our enzyme
assays and eliminated before they reach this screen. Lastly,
compounds showing poor efficacy against the T. gondii-CpIMPDH
parasite may signal problems pertaining to compound uptake,
stability or metabolism. Examples of these varied outcomes are
discussed below.
[0581] A high-content imaging assay to evaluate
anti-cryptosporidial activity of compounds. While the Toxoplasma
model provides outstanding throughput and an excellent first filter
it does not model all aspects of Cryptosporidium biology. A direct
and efficient assay of Cryptosporidium proliferation was also
needed. Fluorescent Vicia villosa lectin (VVL) has been used
previously to score C. parvum growth by fluorescence microscopy.
VVL binds with high specificity to the C. parvum parasite,
labelling sporozoites, intracellular stages, the inner oocyst wall,
but not the outer oocyst wall. To accommodate the increasing number
of compounds entering the SAR pipeline, we adapted the FITC-VVL
immunofluorescence assay to a 96-well plate format and developed an
automated imaging and analysis pipeline. FIG. 58 shows an overview
of the methodology. Plates are fixed, permeabilized and stained
with FITC-VVL and DAPI to numerate parasites and host cells,
respectively. Using a spinning disc microscope, we imaged a X mm
area of each well, providing a robust sample typically consisting
of .about.6000 host cells and .about.2000 parasite stages. The
instrument is programmed to automatically move from well to well,
focus and acquire 20 .mu.M deep image stacks for the entire plate.
A series of automated image compression, manipulation, and
object-finding algorithms was optimized for the recognition of host
cells and parasites using the DAPI and FITC channels. To control
for background staining and biological variability, control wells
are included for background subtraction. The massive data output is
stored, managed and accessed through an Accelrys pipeline database
that performs further statistical analyses and transforms raw
counts into percentage growth relative to a "no drug" control.
[0582] The EC.sub.50 for paromomycin as measured with this assay
was 97 .mu.M (FIG. 58), which isin good agreement with several
previous studies (reported EC.sub.50 ranges from 65-130 .mu.M.
However, at very high concentrations of paromomycin we only detect
.about.70% reduction in parasite number, this may be due to the
labelling of sporozoites that invade the host cell monolayer and
subsequently die or become arrested in development. Interestingly
paromomycin was also found to significantly reduce the mean
parasite area in a dose responsive manner (FIG. 58) and might be
consistent with the parasites present at high paromomycin
concentrations being developmentally arrested. For several of the
test compounds 90% growth inhibition was apparent at the higher
range of concentrations tested, although unlike with paromomycin no
apparent reduction in parasite area was detected (data not shown).
FIG. 58 shows a 2-fold titration of oocysts where the highest
inoculum as 1.2.times.10.sup.6 oocysts per well, for C. parvum
growth assays 5.times.10.sup.5 oocysts were added per well.
[0583] Validation of the fluorescent host cell growth assay. The
differentiation of selective antiparasitic effects from those that
are a secondary consequence of a host cell effect is a critical
issue in drug discovery for intracellular parasites. Cytotoxicity
assays commonly reflect plasma membrane integrity, and are a crude
measure of host cell effects--it is conceivable that more subtle
perturbations of host cell metabolism can have an adverse effect on
parasite proliferation. Therefore we sought to develop a simple and
inexpensive assay of host cell growth. The human ileocecal
adenocarcinoma epithelial cell line, HCT-8, which is commonly used
to maintain C. parvum infection in tissue culture, was engineered
to constitutively express a green fluorescent protein (GFP). Growth
of this cell line was monitored daily using a fluorescent plate
reader. In agreement with previous reports, paromomycin had
negligible effects on the growth of these cells. Sodium butyrate
did inhibit HCT-8 growth in a dose-dependent manner in this assay,
as anticipated due to previous reports of its apoptotic effects in
colonic tumor cell lines. These experiments validate the use of the
fluorescent HCT-8 cell growth assay.
[0584] Identification of highly selective CpIMPDH inhibitors in the
T. gondii model. The antiparasitic activities of 26 compounds from
our medicinal chemistry optimization program were evaluated in the
T. gondii model system. The structures of the compounds are shown
in FIG. 62 alongside a summary of findings. Three compounds, A17
(compound 60), A57 (compound 104) and A66 (102), do not inhibit
CpIMPDH in vitro; as expected, none of these compounds selectively
blocked the growth of T. gondii-CpIMPDH (FIG. 59, FIG. 62). The
remaining compounds inhibit CpIMPDH with values of IC.sub.50
ranging from 9 nM to 2.6 .mu.M. FIG. 59 shows representative data
for fourteen 1,2,3-triazole derivatives in the T. gondii model.
With the exception of A23 (79) and A31 (61), all compounds inhibit
the growth of the T. gondii-CpIMPDH parasite with an
EC.sub.50<10 .mu.M (FIG. 59, 62, 64). Five of the 1,2,3-triazole
derivatives, A100 (23), A102 (25), A103 (26), A109 (32) and A110
(33), exhibit selectivity .gtoreq.36-fold for the T. gondii-CpIMPDH
parasite over wild-type T. gondii (FIG. 59). Therefore, the
antiparasitic effects of these compounds can be confidently
attributed to the inhibition of CpIMPDH. A99 (22) is
.gtoreq.17-fold selective and the remaining compounds range in
selectivity from 0.9-14-fold (FIG. 59). All compounds have similar
effects on both wild-type and T. gondii-.DELTA.HXGPRT parasites
(FIG. 59), indicating that the lack of selectivity derives from
off-target effects unrelated to TgIMPDH.
[0585] Surprisingly, initially we did not observe a significant
correlation between the potency of a compound in the enzyme assay
and inhibition of T. gondii-CpIMPDH proliferation (FIG. 60). We
wondered if this may be an issue of bioavailability and linked to
the presence of serum in the parasite growth medium. To test this
hypothesis, enzyme inhibition was also evaluated in the presence of
BSA. A strong positive correlation is observed between inhibition
of T. gondii-CpIMPDH proliferation and potency of CpIMPDH enzyme
inhibition in the presence of BSA (FIG. 60; r=-0.94, p<0.0001).
Selectivity in the T. gondii model also correlates well with the
potency of enzyme inhibition in the presence of BSA (FIG. 60;
r=-0.92, p<0.0001). These observations indicate that the
IC.sub.50 value in the presence of BSA is a useful proxy for
antiparasitic activity modelled by T. gondii-CpIMPDH
proliferation.
[0586] The T. gondii model and predicting off-target host cell
effects. Host cell growth was also assayed to assess the
contribution of host cell effects to antiparasitic activity (FIGS.
59, 62, and 64). In general, strong host cell effects are observed
in compounds that display little selectivity in the T. gondii model
(FIGS. 59, 62, and 64). With three exceptions (A98, A99, and A108),
compounds that inhibited the proliferation of wild-type T. gondii
with EC.sub.50<10 .mu.M also inhibited the proliferation of host
cells. Three compounds (A82, A90, and A105) display little
selectivity in the T. gondii model and do not inhibit host cell
growth, suggesting that the antiparasitic activities of A82, A90,
and A105 do not result from the inhibition of CpIMPDH or TgIMPDH.
Instead, A82, A90, and A105 may act on other T. gondii targets not
present in the host cell. Conversely, A100, A102, and A103 have
EC.sub.50>20 .mu.M against wild-type T. gondii yet inhibit HCT-8
cell growth significantly at 12.5 .mu.M and 25 .mu.M (FIG. 62).
[0587] CpIMPDH inhibitors with significantly improved
anti-cryptosporidial activity. The high-content imaging assay was
used to evaluate the anti-cryptosporidial activity of the
1,2,3-triazole CpIMPDH inhibitors at 12.5 .mu.M and 25 .mu.M (FIG.
59). All compounds inhibited C. parvum growth by at least 48% at a
concentration of 25 .mu.M (FIG. 59) and thus had equal or markedly
improved anticryptosporidial efficacy when compared to parent
compound A (53) (EC.sub.50 25 .mu.M-50 .mu.M). Unlike paromomycin,
a significant reduction in parasite area was not detected (data not
shown). The average area of the host cell nucleus was also recorded
as a potential indicator of host cell cytotoxicity and likewise no
significant change in host cell nuclei size was detected (data not
shown). Encouragingly there was a negative trend between
anticryptosporidial activity and host cell growth inhibition (data
not shown), indicating that improvements in anticryptosporidial
activity are not coincident with secondary effects on the host
cell.
[0588] To provide quantitative data to the SAR pipeline, the values
of EC.sub.50 were determined for seven compounds. A82 (7), A90
(14), A92 (16), A98 (21) and A105 (28) had EC.sub.50 values between
3 .mu.M and 13 .mu.M (FIG. 62). Compounds A103 (26) and A110 (33)
were found to be potent inhibitors of C. parvum growth with
EC.sub.50 values of <0.8 .mu.M (FIG. 61). As shown above, A103
and A110 also have negligible effects on host cell growth and
exhibit good selectivity in the T. gondii model. Therefore, A103
and A110 have improved specificity, improved efficacy and a good
therapeutic window.
[0589] While not every compound that showed activity against the T.
gondii-CpIMPDH parasite had strong anticryptosporidial activity,
none of the compounds showing poor activity in the T.
gondii-CpIMPDH model display significant anticryptosporidial
activity. The T. gondii assay also immediately flagged compounds
with poor bioavailability and those that showed parasite killing
due to off-target effects. We conclude that the T. gondii-CpIMPDH
model provides valuable information regarding compound specificity
and is a fast and highly informative filter for compound
progression through medicinal chemistry optimization.
[0590] Discussion
[0591] Despite the tremendous public health impact of
cryptosporidiosis efforts to develop new and more effective
treatments for this diseases have been languishing. There are a
number of reasons for this, but lack of suitable tissue culture and
animal models to assess drug candidates is currently the most
prominent roadblock. To overcome this challenge we have developed a
facile screening pipeline to evaluate the antiparasitic activity of
CpIMPDH inhibitors. The backbone of this pipeline is provided by a
T. gondii model parasite that mirrors Cryptosporidium purine
nucleotide pathways and depends on CpIMPDH. The T. gondii model
reliably eliminates compounds from further consideration and
provides a useful filter to identify off-target activities.
However, efficacy in the T. gondii model does not always guarantee
anti-cryptosporidial activity. This disparity likely arises from
fundamental differences in the biology of the two parasites. T.
gondii and C. parvum infect different tissues, and occupy different
intracellular compartments. The parasitophorous membrane of T.
gondii is in direct contact with the host cell cytoplasm. In
contrast, C. parvum remains beneath the apical membrane of the host
cell and is considered `extracytoplasmic` due to the presence of a
parasite induced host cell actin patch along with other peculiar
and still largely uncharacterized structures including a dense band
visible in electron micrographs. This band separates the parasite's
parasitophorous vacuole from the host cell cytoplasm and has been
hypothesized to be involved in drug and nutrient uptake.
Furthermore, the two parasites, and their respective host cells,
have different repertoires of drug efflux transporters, which can
also account for the differences in inhibitor sensitivity. While
the T. gondii assay does not fully negate the necessity of testing
in Cryptosporidium directly, it has proven indispensable to winnow
candidate compounds to a manageable number amenable to this more
challenging model. We have used the pipeline to identify two
promising candidates for anticryptosporidial chemotherapy: A103 and
A110. These compounds are >100.times. more potent than
paromomycin, the current standard for anticryptosporidial
activity.
[0592] Supplemental Experimental Data
[0593] In vitro culture of Cryptosporidium parvum. The human
ileocecal adenocarcinoma epithelial cell line, HCT-8, was used to
support C. parvum infection in vitro. HCT-8 cells were maintained
in RPMI-1640 (Hyclone) supplemented with 10% FBS, 1 mM sodium
pyruvate, 50 U/m penicillin, 50 .mu.g/mL streptomycin, and
amphotericin B. Cryptosporidium parvum oocysts were a kind gift
from either Dr. Mead (Emory University) of Dr Kissinger (University
of Georgia). Purified oocysts were received in 2% potassium
dichromate and stored at 4.degree. C. for up to 4 months.
[0594] For the HCl assay the day prior to infection 200 000 HCT-8
cells were seeded into black, optical quality, thin bottom, 96-well
plates (DB Falcom) to achieve a 70% confluent monolayer on the day
of infection. To facilitate oocyst excystation a procedure
described by Gut et al., was followed. Briefly, oocysts were washed
twice with 1 mL of PBS (pH7.2), incubated for 10 minutes at
37.degree. C. in 1 mL 10 mM of HCl and then incubated for a further
10 minutes in 0.2 mL of 200 .mu.M sodium taurocholate at 15.degree.
C. This oocyst suspension was diluted directly with DMEM (Hyclone)
supplemented with 2% FBS, 50 U/m penicillin, 50 .mu.g/mL
streptomycin, amphotericin B and 0.2 mM L-glutamine (infection
medium) to inoculate host cell monolayers at 5.times.10.sup.5
oocysts per well. Oocysts were cultured on host cell monolayers for
3 hours at 37.degree. C. Unexcysted oocysts and oocyst walls were
then removed by aspiration and each well washed with 0.2 mL PBS
(pH7.2). Infection medium was then added to the monolayers and
infection was allowed to progress for 48 hours.
[0595] Vicia villosa lectin (VVL) immunofluorescence assay and High
Content Imaging. The VVL IFA was performed in a 96-well format as
follows. Following 48 hours of culture C. parvum infected HCT-8
monolayers were washed with 0.2 mL/well PBS and the monolayer was
fixed with 0.2 mL/well of 3% paraformaldehyde/PBS, permeabilized
with 0.25% Triton-X-100/PBS and blocked with 4% BSA/PBS. When
necessary plates were stored at 4.degree. C. for up to 2 weeks 0.1
mL of fluorescein (FITC)-conjugated VVL (Vector Labs) at 0.5
.mu.g/mL in 1% BSA/PBS was applied to wells and incubated for 45
minutes. The plates were washed twice with 200 .mu.L/well of PBS,
in the first wash DAPI at 0.1 .mu.g/mL was included. Finally 200
.mu.L/well of PBS was added to the plates prior to storage at
4.degree. C. protected from light.
[0596] Following the labelling of the C. parvum infected HCT-8 cell
monolayer with FITC-VVL and DAPI, confocal images were acquired
using a scanning microscope (BD Biosciences Bioimager P435).
[0597] Montage images of 9-16, 40.times. fields per well
automatically focused, captured, compressed and saved. Images from
each plate were analyzed using object-finding algorithms
(Attovision Software, BD Biosciences) optimized for the recognition
of FITC-VVL labelled C. parvum parasites or DAPI labelled host cell
nuclei. The object finding analysis step recorded the number and
area of objects per montage image, the output files and a plate map
file were then passed onto an automated analysis pipeline (Pipeline
Pilot Software, Accelrys) to calculate, the mean number of
parasites, the ratio of parasite number to host cell nuclei number,
the mean area of parasites and host cell nuclei by well and
percentage growth by treatment as compared to the no drug control.
These analyses were output in graphical format in one PDF file per
plate and the numerical values tabulated in html format.
[0598] All test compounds were stored as 0.1 M stocks in DMSO at
-20.degree. C. and further diluted in DMSO to a 200.times. working
stocks for each dilution, such that the final concentration of DMSO
in the infection medium was 0.5%. For the no drug control DMSO
alone was added to triplicate wells. As a control for C. parvum
growth a high paromomycin concentration (0.8 mg/mL) was included on
each plate in triplicate wells. Plates where this paromomycin
control did not inhibit 70-80% of parasite growth were manually
inspected to confirm appropriate imaging and analysis. Plates were
omitted from final analysis where it was apparent that a lack of
inhibition was due to poor parasite growth.
[0599] Fluorescent HCT-8 host cell growth assay. HCT-8 cells were
transfected with the pmaxGFP plasmid (Amaxa) using Lipofectamine
(Invitrogen) following the manufactures instruction. Fluorescent
lines were then selected and cloned using FACS. Confluent
monolayers of pmaxGFP expressing cells were harvested from T75
flasks and passed through a 40 .mu.m cell strainer. Cells were then
seeded at 4000 cells per well in a volume of 200 .mu.L into black,
optical quality, thin bottom, 96-well plates (DB Falcon). All test
compounds were diluted in DMSO to prepare a 200.times. working
stock for each dilution. Appropriate wells were spiked with 1 .mu.l
such that the final concentration of DMSO was 0.5%. The
fluorescence was read daily with a SpectraMax M22/M2e (Molecular
Devices) plate reader (Ex 485, Em 530) for 6-7 days. The percent
inhibition was calculated on a day within the exponential phase of
growth.
Example 39
Structural Determinants of Inhibitor Selectivity in Prokaryotic IMP
Dehydrogenases
[0600] The protozoan parasite Cryptosporidium parvum is a major
cause of gastrointestinal disease; no effective drug treatment
exists to treat this infection. Curiously, CpIMPDH is most closely
related to prokaryotic IMPDHs, suggesting that the parasite
obtained its IMPDH gene via horizontal transfer. We previously
identified inhibitors of CpIMPDH that do not inhibit human IMPDHs.
Here we show that these compounds also inhibit IMPDHs from
Helicobacter pylori, Borrelia burgdorferi, and Streptococcus
pyogenes, but not IMPDHs from Escherichia coli, Tritrichomonas
foetus and Leishmania donovani. Importantly, a second generation
inhibitor blocks H. pylori growth. The presence of Ala165 and
Tyr358 comprise a structural motif that defines susceptible
enzymes, as verified by site-directed mutagenesis of E. coli IMPDH.
We propose that IMPDH-targeted inhibitors represent a new class of
antibiotics for treatment of a wide variety of pathogenic bacteria,
including extensively drug resistant strains.
[0601] In this study, we explore the possibility that the
inhibitors of the CpIMPDH might be broad spectrum inhibitors of
prokaryotic IMPDHs. We find that IMPDHs from H. pylori (the
causative agent of gastric ulcer/stomach cancer), Borrelia
burgdorferi (the causative agent of Lyme disease) and Streptococcus
pyogenes (a major cause of nosocomial infections) are inhibited by
these compounds while Escherichia coli IMPDH is resistant.
Importantly, a second generation CpIMPDH inhibitor blocks H. pylori
growth, demonstrating that these compounds have antibacterial
activity. A structural motif is identified that defines susceptible
enzymes; this motif is found in a wide variety of pathogenic
bacteria. These observations suggest that IMPDH-targeted inhibitors
can be developed into a new class of moderate-spectrum
antibiotics.
Results and Discussion
[0602] Expression, Purification and Characterization of Recombinant
IMPDHs. We expressed and purified prokaryotic IMPDHs from
representative organisms: H. pylori (Gram negative .epsilon.
proteobacteria), E. coli (Gram negative .gamma. proteobacteria), B.
burgdorferi (spirochete), S. pyogenes (Gram positive) and the
protozoan parasite T. foetus, which also appears to have obtained
its IMPDH gene from a prokaryote. We also expressed an additional
eukaryotic IMPDH from the protozoan parasite L. donovani. CpIMPDH
is most closely related to HpIMPDH (FIG. 68), but also has
.about.50% sequence identity to EcIMPDH, BbIMPDH and SpIMPDH.
Sequence identity drops to 32% for TfIMPDH, which is comparable to
that of the eukaryotic enzymes. EcIMPDH, BbIMPDH, TfIMPDH, SpIMPDH
and LdIMPDH have been characterized previously. The kinetic
parameters of HpIMPDH are very similar to those of CpIMPDH, and are
generally characteristic of bacterial IMPDHs. Importantly,
structures are available for TfIMPDH, SpIMPDH and BbIMPDH as well
as for CpIMPDH and the human enzymes.
[0603] Spectrum of inhibition of CpIMPDH inhibitors. Compounds A-H
also inhibit HpIMPDH, the enzyme most similar to CpIMPDH. With the
exception of G, all of the compounds have similar potency for both
enzymes, with values of IC.sub.50 ranging from 0.6 to 5 .mu.M. A-H
are noncompetitive (mixed) inhibitors of HpIMPDH with respect to
NAD.sup.+ (data not shown), as observed with CpIMPDH. Importantly,
the values of K.sub.i and IC.sub.50 are similar, as expected for
noncompetitive inhibition. These observations suggest that the
inhibitor binding sites are similar on both HpIMPDH and
CpIMPDH.
[0604] The compounds also inhibit BbIMPDH with similar potency to
CpIMPDH and HpIMPDH (FIG. 67). However, whereas G and H are
submicromolar inhibitors of SpIMPDH, A-F are markedly less
effective against this enzyme, with IC.sub.50 values ranging from
13 to 90 .mu.M. No inhibition of EcIMPDH and TfIMPDH is observed at
100 indicating that the values of IC.sub.50 for A-H must be
>1000 .mu.M. This result is especially surprising for EcIMPDH
because this enzyme has the same overall similarity to CpIMPDH as
the sensitive enzymes. As expected for a eukaryotic IMPDH, A-H do
not inhibit LdIMPDH.
[0605] Inhibition of H. pylori growth. H. pylori is cultured in a
nutrient rich medium (Brucella broth), which provides a stringent
test for the antibiotic potential of IMPDH-targeted inhibitors. H.
pylori contains a gene encoding xanthine/guanine
phosphoribosyltransferase, suggesting that these bacteria can
salvage xanthine and guanine from the media. If this salvage
pathway is efficient, H. pylori will be resistant to IMPDH
inhibitors. Therefore we investigated the sensitivity of H. pylori
to a second generation CpIMPDH inhibitor, C91 (FIG. 41). C91
inhibits HpIMPDH with an IC.sub.50=25.+-.3 nM, which is comparable
to that observed for inhibition of CpIMPDH (IC.sub.50=8.+-.3 nM;
[13]).
[0606] FIG. 69 shows that 20 .mu.M C91 is sufficient to block the
proliferation of a H. pylori culture exiting stationary phase.
Higher concentrations of C91 display bacteriocidal effects, with
only 23% of the colony forming units remaining after 24 hr
treatment with 200 .mu.M. Exponentially growing H. pylori cells are
also sensitive to C91; a concentration of 60 .mu.M is sufficient to
block growth while higher concentrations are bacteriocidal.
[0607] Structural determinants of inhibitor susceptibility. The
structure of CpIMPDH with the second generation inhibitor C64
identifies a possible binding site for the inhibitors A-H (FIGS. 70
and 71). IMPDH is a tetramer; surprisingly, C64 binds across a
dimer interface, bending around Ala165 and stacking with Tyr358.
Both Ala165 and Tyr358 are conserved in the sensitive enzymes, but
diverged in the resistant enzymes, suggesting that these residues
determine susceptibility to the C series inhibitors, and possibly
the other compounds.
[0608] To determine the role of Ala165 and Tyr358 in defining the
inhibitor susceptibility, we replaced the corresponding residues of
the resistant EcIMPDH with their CpIMPDH counterparts to create
three variants: S250A, L444Y and S250A/L444Y. The steady-state
kinetic parameters of S250A and S250A/L444Y are comparable to those
of wild-type EcIMPDH, though the value of K.sub.m for NAD.sup.+ is
increased by more than 5-fold in L444Y. Unlike EcIMPDH, significant
inhibition is observed when the S250A and L444Y variants are
incubated with 100 .mu.M A-H, suggesting that the single mutations
increase sensitivity by factors of at least 2-10 (unfortunately,
the solubility of the compounds does not permit the values of
IC.sub.50 to be determined). In contrast, A-H are potent inhibitors
of the S250A/L444Y enzyme, with values of IC.sub.50 comparable to
CpIMPDH (FIG. 67). These observations indicate that together Ala165
and Tyr358 define the structural motif required for susceptibility
to A-H.
[0609] The conformational contribution to inhibitor selectivity. As
noted in the introduction, IMPDH undergoes a conformational change
in the middle of its catalytic cycle that brings a mobile flap into
the NAD site (FIG. 68). The competition of the flap for this site
can be an important determinant of inhibitor susceptibility, and
might explain the low susceptibility of SpIMPDH despite the
presence of Ala165 and Tyr358 (FIG. 67). Therefore we determined
the equilibrium between open and closed conformations (K.sub.c)
using a multiple inhibitor experiment.
[0610] Tiazofurin inhibition illustrates the magnitude of the
conformational contribution to inhibitor selectivity. The
tiazofurin binding site is conserved among prokaryotic IMPDHs,
which predicts that CpIMPDH, HpIMPDH, BbIMPDH, SpIMPDH, EcIMPDH and
TfIMPDH should all bind tiazofurin with similar affinity, yet the
values of K.sub.i vary from 1-69 mM. When the observed values are
adjusted for competition from the mobile flap, the resulting
"intrinsic values" are indeed nearly identical, ranging from
0.3-0.7 mM. In contrast, the intrinsic values of K.sub.i for ADP
range from 0.2-9 mM, reflecting the structural divergence of the
ADP binding sites.
[0611] The intrinsic affinities of A-H. We determined the intrinsic
values of IC.sub.50 for A-H in order to assess how competition with
the mobile flap contributes to susceptibility (FIG. 67). Inspection
of the intrinsic values of IC.sub.50 reveals two distinct inhibitor
binding modes. The intrinsic values of IC.sub.50 of C range between
0.18-0.36 .mu.M for CpIMPDH, HpIMPDH, BbIMPDH and S250A/L444Y (FIG.
67), reflecting the conservation of this binding site. Likewise,
the intrinsic affinities of compounds A, B, D, E and F are within a
factor of 2 for all four enzymes, indicating that the binding sites
of these compounds are also conserved. These observations suggest
that compounds A, B, D, E and F most likely occupy the same binding
site as C.
[0612] In contrast, the intrinsic values of A-F for SpIMPDH are
very different from CpIMDPH, indicating that this binding site is
significantly different in SpIMPDH. Only one substitution is
present within 3.5 .ANG. of C64: Met326 is a Leu in SpIMPDH.
However, the Leu substitution is also present in HpIMPDH and
BbIMPDH, and therefore cannot account for the different
susceptibility. The next nearest substitution is Thr for Ser164;
the side chain of Ser164 is 5 .ANG. away from C64, but might be
closer to the A, B and D-F.
[0613] A very different trend is observed in the intrinsic
affinities of G and H. SpIMPDH is most similar to CpIMPDH, while
HpIMPDH and BbIMPDH display lower affinities for these compounds
(FIG. 67). These observations suggest that G/H bind in a region
that is conserved in SpIMPDH and CpIMPDH, but different in the
other enzymes. Therefore, at least a portion of the G/H binding
site must be distinct from the site that binds A-F.
[0614] Implications for the design of antibiotics targeting IMPDH.
The above findings indicate that Ala165 and Tyr358 comprise a
structural motif that defines enzymes susceptible to CpIMPDH
inhibitors. A BLAST search reveals that these critical residues are
present in IMPDHs from a wide variety of pathogenic bacteria in
addition to C. parvum, B. burgdorferi and H. pylori: Campylobacter
lari (food poisoning), Campylobacter jejuni (food poisoning),
Arcobacter butzleri (food poisoning), Bacteroides capillosis
(abscesses), Fusobacterium nucleatum (periodontitis, Lemierre's
syndrome, skin ulcers), Burkholderia cenocepacia (infection in
Cystic Fibrosis), S. pneumoniae (pneumonia), Clostridia botulinum
(botulism), Neisseria gonorrhoeae (gonorrhea), Mycobacterium
tuberculosis (tuberculosis), M. leprae (leprosy), Neisseria
meningitides (bacterial meningitis), Staphylococcus aureus (major
cause of nosocomial infection), Acinetobacter baumannii (wound
infection), Bacillus anthracis (anthrax) and Clostridium botulinum.
Importantly, several of these pathogens have developed multi-drug
resistant strains, so new antibiotics are urgently needed. Our
results suggest that IMPDH inhibition provides a promising strategy
for the development of a new moderate spectrum antibiotic.
Prokaryotic-specific inhibitors such as C91 will be invaluable in
validating IMPDH as a target for antibiotic chemotherapy.
Significance
[0615] The rising tide of antibiotic resistance creates an urgent
need for new drugs to treat bacterial infections, but years of
neglect have depleted the antibiotic pipeline. The re-purposing of
other drug development programs for antibiotic discovery is a
promising strategy to address this problem. Inosine
5'-monophosphate dehydrogenase (IMPDH), a key enzyme in the
biosynthesis of the precursors for RNA and DNA, presents an
intriguing opportunity for such re-purposing. IMPDH is a promising
target for drugs against the protozoan parasite Cryptosporidium
parvum, a major cause of diarrhea and malnutrition and a potential
bioterrorism agent. Curiously, CpIMPDH is most closely related to
prokaryotic IMPDHs, suggesting that the parasite obtained its IMPDH
gene via horizontal transfer. We previously identified inhibitors
of CpIMPDH that do not inhibit human IMPDHs. Here we show that
selective inhibitors of CpIMPDH also inhibit IMPDHs from the
pathogenic bacteria Helicobacter pylori, Borrelia burgdorferi, and
Streptococcus pyogenes. Importantly, a second generation CpIMPDH
inhibitor blocks H. pylori growth in rich media, demonstrating that
these compounds have antibacterial activity. Importantly,
susceptible enzymes are defined by a structural motif that is found
in IMPDHs from a wide variety of pathogenic bacteria, suggesting
that IMPDH-targeted inhibitors can be developed into a new class of
moderate spectrum antibiotics.
Experimental Procedures
[0616] Materials. Compounds D, E, F, G, and H were purchased from
ChemDiv Inc. (San Diego, Calif.), Compounds A, B and C were
synthesized as described previously. Compound C91 was synthesized
as described. All other chemicals were obtained from Fisher
Scientific, unless mentioned otherwise. Plasmid containing the guaB
gene of S. pyogenes was a generous gift of Dr. Cameron Ashbaugh. H.
pylori total genomic DNA was obtained from American Type Culture
Collection (ATCC). L. donovani IMPDH coding sequence was the gift
of Dr. Buddy Ullman.
[0617] Enzyme Cloning and Purification. Recombinant T. foetus, B.
burgdorferi, E. coli and C. parvum IMPDH were expressed in guaB
strains of E. coli (which lack endogenous IMPDH) and purified as
described previously. The S250A, L444Y and S250A/L444Y mutants of
E. coli IMPDH were constructed using Quikchange (Stratagene, La
Jolla, Calif.). Enzymes were expressed and purified as previously
described.
[0618] To express L. donovani IMPDH, a NcoI site was created at the
beginning of the LdIMPDH coding sequence and the NcoI-PstI fragment
was cloned into pKK233-2 to create the plasmid pLDI, which
expresses LdIMPDH under control of the trc promoter. Cultures were
induced with 0.5 mM IPTG and grown overnight. Cells were harvested
by centrifugation, resuspended in Buffer A, lysed by sonication and
clarified by centrifugation followed by filtration through a 45
.mu.m cellulose acetate filter. Protein was applied to a Poros HS
strong cation exchange resin (PerSeptive Biosystems)
pre-equilibrated with 20 mM NaP.sub.i, pH 7.5, 1 mM DTT (Buffer B).
LdIMPDH was eluted with a gradient of 0-0.9 M NaCl. Fractions
containing IMPDH activity were pooled and applied to IMP affinity
resin. The column was washed with Buffer B and enzyme was eluted
with Buffer B containing 0.5 M KCl, 1 mM IMP. The specific activity
of the final preparation was 2.6 .mu.moles/min-mg.
[0619] The H. pylori and S. pyogenes guaB genes were cloned into
pET28a with 6.times. His-tags. Bacteria were grown at 30.degree. C.
in LB medium containing 25 .mu.g/mL kanamycin until the OD.sub.600
reached approximately 0.6. Expression was initiated by the addition
of 0.5 mM IPTG and the temperature was changed to 25.degree. C.
Bacteria were harvested after 16 hours. The cell pellet was rinsed
(3.times.) with 50 mM phosphate buffer, 500 mM NaCl, 5 mM
imidazole, pH 8.0, 1 mM IMP and 5 mM .beta.-mercaptoethanol, and
lysed by sonication. The lysate was clarified by centrifugation and
loaded on a Ni-NTA column (Qiagen). The purified protein were
eluted in 50 mM phosphate buffer, 500 mM NaCl, 250 mM imidazole, pH
8.0, 1 mM IMP and 5 mM .beta.-mercaptoethanol, concentrated and
dialyzed against 50 mM Tris-HCl, pH 8.0, and 10% glycerol. The
protein concentration was determined by using Bradford dye
procedure (BioRad).
[0620] Steady State Enzyme Kinetics: IMPDH assays were performed in
50 mM Tris-HCl, pH 8.0, 100 mM KCl, 3 mM EDTA and 1 mM DTT.
Activity was routinely assayed in the presence of 50 nM IMPDH at
25.degree. C. NADH production was monitored either by following
absorbance change at 340 nm using a Hitachi U-2000
spectrophotometer (.epsilon.=6.2 mM.sup.-1 cm.sup.-1). IMPDHs are
prone to NAD.sup.+substrate inhibition. Therefore the steady state
kinetics for HpIMPDH were initially analyzed by varying NAD.sup.+at
saturating IMP concentrations to determine the value of K.sub.m for
NAD.sup.+, then by varying IMP at the fixed NAD.sup.+concentration
as close to saturating as practical. Using the SigmaPlot program
(SPSS, Inc.), initial velocity data were fit to the
Michaelis-Menten equation (Equation a) and/or the uncompetitive
substrate inhibition equation (Equation b), as follows,
.nu.=V.sub.m[S]/(K.sub.m+[S]) (a)
.nu.=V.sub.m/(1+K.sub.m/[S]+[S].sup.2/K.sub.ii) (b)
where .nu. represents the velocity, V.sub.m is the maximal
velocity, S is the substrate concentration, K.sub.m is the
Michaelis constant, K.sub.ii is the intercept inhibition constant
(X-Y). The values of k.sub.cat determined under both conditions are
in good agreement.
[0621] Inhibitor Kinetics. Enzyme was incubated with inhibitor (50
pM-100 .mu.M) for 10 min at room temperature prior to addition of
substrates. IC.sub.50 values were calculated for each inhibitor
according to Equation c using the SigmaPlot program (SPSS,
Inc.):
.upsilon..sub.i=.upsilon..sub.0/(1+[I]/IC.sub.50) (c)
where .upsilon..sub.i is initial velocity in the presence of
inhibitor (I) and .upsilon..sub.0 is the initial velocity in the
absence of inhibitor.
[0622] Assays were carried out in assay buffer at 25.degree. C.
with 50 nM IMPDH and NADH production was monitored by following
fluorescence. The values of K.sub.i with respect to NAD.sup.+ were
determined by using fixed concentrations of IMP and varied
NAD.sup.+ concentrations. Data were fitted according to Equation d
(noncompetitive inhibition) using SigmaPlot program (SPSS,
Inc.):
.upsilon.=V.sub.m[S]/{K.sub.m(1+[I]/K.sub.is)+[S](1+[I]/K.sub.ii)}
(d)
where K.sub.ii and K.sub.is represent the intercept and slope
inhibition constants, respectively. The best fits were determined
by the relative fit error.
[0623] Multiple Inhibitor Kinetics. Multiple inhibitor experiments
with tiazofurin and ADP were performed at constant IMP and
NAD.sup.+ (see Table S3 for concentrations). Initial velocities
were fit to equation e using SigmaPlot:
.nu.=.nu..sub.0/[1+[I]/K.sub.i+[J]/K.sub.j+[I][J]/.alpha.K.sub.iK.sub.j]
(e)
where .nu. is the initial velocity, .nu..sub.0 is the initial
velocity in the absence of inhibitor, K.sub.i and K.sub.j are the
inhibition constants for the inhibitors I and J, respectively and
.alpha. is the interaction constant. In wild-type IMPDH, the
tiazofurin and ADP are strongly synergistic inhibitors with an
interaction constant .alpha.=0.007. This observation suggests that
one inhibitor shifts the enzyme into the open conformation, thus
promoting the association of the second inhibitor. Further, the
value of .alpha. approximates the fraction of enzyme in the open
conformation, so the value of K.sub.c can be obtained:
K.sub.c=(1-.alpha.)/.alpha. (f)
[0624] H. pylori growth assays. A stationary culture of H. pylori
strain G27 `Merrell` was diluted into Brucella broth with fresh 10%
fetal bovine serum to an OD.sub.600=0.025 (.about.10.sup.4 colony
forming units/.mu.l). Cultures (200 .mu.l) were incubated with C91
added in 2 .mu.l aliquots of DMSO solution, or 2 .mu.l of DMSO
alone, for 24 hrs. Colony forming units were determined.
Alternatively, exponentially growing cultures were diluted to
.about.10.sup.4 colony forming units/.mu.l and treated as
described.
INCORPORATION BY REFERENCE
[0625] All of the U.S. patents and U.S. published patent
applications cited herein are hereby incorporated by reference.
EQUIVALENTS
[0626] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *